IL-3 variant hematopoiesis fusion protein

ABSTRACT

The present invention relates to fusion molecules composed of human interleukin-3 (hIL-3) variant or mutant proteins (muteins) functionally joined to a second colony stimulating factor (CSF), cytokine, lymphokine, interleukin or IL-3 variant. These hIL-3 variants contain amino acid substitutions and may also have amino acid deletions at both the N- and C-termini. The invention also relates to pharmaceutical compositions containing the fusion molecules and methods for using them.

This a divisional of Ser. No. 08/192,299 which was filed Feb. 4, 1994now U.S. Pat. No. 5,738,849; which is a continuation-in-part of U.S.Ser. No. 08/411,796 now U.S. Pat. No. 5,677,149; which was filed asinternational application PCT/US93/11198 on Nov. 22, 1993, which enteredthe U.S. national stage under 35 U.S.C. § 371 on Apr. 6, 1995; andPCT/US93/11198, which is a continuation-in-part of U.S. Ser. No.07/981,044; filed Nov. 24, 1992 which is now abandoned.

FIELD OF THE INVENTION

The present invention relates to fusion molecules composed of mutants orvariants of human interleukin-3 (hIL-3) fused to a second colonystimulating factor (CSF), cytokine, lymphokine, interleukin,hematopoietic growth factor or IL-3 variant with or without a linker

BACKGROUND OF THE INVENTION

Colony stimulating factors (CSFs) which stimulate the differentiationand/or proliferation of bone marrow cells have generated much interestbecause of their therapeutic potential for restoring depressed levels ofhematopoietic stem cell-derived cells. CSFs in both human and murinesystems have been identified and distinguished according to theiractivities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF(M-CSF) stimulate the in vitro formation of neutrophilic granulocyte andmacrophage colonies, respectively while GM-CSF and interleukin-3 (IL-3)have broader activities and stimulate the formation of both macrophage,neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulatesthe formation of mast, megakaryocyte and pure and mixed erythroidcolonies (when erythropoietin is added in combination).

Because of its ability to stimulate the proliferation of a number ofdifferent cell types and to support the growth and proliferation ofprogenitor cells, IL-3 has potential for therapeutic use in restoringhematopoietic cells to normal amounts in those cases where the number ofcells has been reduced due to diseases or to therapeutic treatments suchas radiation and chemotherapy.

Interleukin-3 (IL-3) is a hematopoietic growth factor which has theproperty of being able to promote the survival, growth anddifferentiation of hematopoietic cells. Among the biological propertiesof IL-3 are the ability (a) to support the growth and differentiation ofprogenitor cells committed to all, or virtually all, blood celllineages; (b) to interact with early multipotential stem cells; (c) tosustain the growth of pluripotent precursor cells; (d) to stimulateproliferation of chronic myelogenous leukemia (CML) cells; (e) tostimulate proliferation of mast cells, eosinophils and basophils; (f) tostimulate DNA synthesis by human acute myelogenous leukemia (AML) cells;(g) to prime cells for production of leukotrienes and histamines; (h) toinduce leukocyte chemotaxis; and (i) to induce cell surface moleculesneeded for leukocyte adhesion.

Mature human interleukin-3 (hIL-3) consists of 133 amino acids. It hasone disulfide bridge and two potential glycosylation sites (Yang, etal., CELL 47:3 (1986)).

Murine IL-3 (mIL-3) was first identified by Ihle, et al., J. IMMUNOL.126:2184 (1981) as a factor which induced expression of a T cellassociated enzyme, 20-hydroxysteroid dehydrogenase. The factor waspurified to homogeneity and shown to regulate the growth anddifferentiation of numerous subclasses of early hematopoietic andlymphoid progenitor cells.

In 1984, cDNA clones coding for murine IL-3 were isolated (Fung, et al.,NATURE 307:233 (1984) and Yokota, et al., PROC. NATL. ACAD. SCI. USA81:1070 (1984)). The murine DNA sequence coded for a polypeptide of 166amino acids including a putative signal peptide.

The gibbon IL-3 sequence was obtained using a gibbon cDNA expressionlibrary. The gibbon IL-3 sequence was then used as a probe against ahuman genomic library to obtain a human IL-3 sequence.

Gibbon and human genomic DNA homologues of the murine IL-3 sequence weredisclosed by Yang, et al., CELL 47:3 (1986). The human sequence reportedby Yang, et al. included a serine residue at position 8 of the matureprotein sequence. Following this finding, others reported isolation ofPro⁸ hIL-3 cDNAs having proline at position 8 of the protein sequence.Thus it appears that there may be two allelic forms of hIL-3.

Dorssers, et al., GENE 55:115 (1987), found a clone from a human cDNAlibrary which hybridized with mIL-3. This hybridization was the resultof the high degree of homology between the 3′ noncoding regions of mIL-3and hIL-3. This cDNA coded for an hIL-3 (Pro⁸) sequence.

U.S. Pat. No. 4,959,455 and U.S. disclose human IL-3 and gibbon IL-3cDNAs and the protein sequences for which they code. The hIL-3 disclosedhas serine rather than proline at position 8 in the protein sequence.

Clark-Lewis, et al., SCIENCE 231:134 (1986) performed a functionalanalysis of murine IL-3 analogues synthesized with an automated peptidesynthesizer. The authors concluded that the stable tertiary structure ofthe complete molecule was required for full activity. A study on therole of the disulfide bridges showed that replacement of all fourcysteines by alanine gave a molecule with 1/500th the activity as thenative molecule. Replacement of two of the four Cys residues byAla(Cys⁷⁹, Cys¹⁴⁰->Ala⁷⁹, Ala¹⁴⁰) resulted in an increased activity. Theauthors concluded that in murine IL-3 a single disulfide bridge isrequired between cysteines 17 and 80 to get biological activity thatapproximates physiological levels and that this structure probablystabilizes the tertiary structure of the protein to give a conformationthat is optimal for function. (Clark-Lewis, et al., PROC. NATL. ACAD.SCI. USA 85:7897 (1988)).

International Patent Application (PCT) WO 88/00598 discloses gibbon- andhuman-like IL-3. The hIL-3 contains a Ser⁸->Pro⁸ replacement.Suggestions are made to replace Cys by Ser, thereby breaking thedisulfide bridge, and to replace one or more amino acids at theglycosylation sites.

EP-A-0275598 (WO 88/04691) illustrates that Ala¹ can be deleted whileretaining biological activity. Some mutant hIL-3 sequences are provided,e.g., two double mutants, Ala¹->Asp¹, Trp¹³->Arg¹³ (pGB/IL-302) andAla¹->Asp¹, Met³->Thr³ (pGB/IL-304) and one triple mutant Ala¹->Asp¹,Leu⁹->Pro⁹, Trp¹³->Arg¹³ (pGB/IL-303).

WO 88/05469 describes how deglycosylation mutants can be obtained andsuggests mutants of Arg⁵⁴Arg⁵⁵ and Arg¹⁰⁸Arg¹⁰⁹Lys¹¹⁰ might avoidproteolysis upon expression in Saccharomyces cerevisiae by KEX2protease. No mutated proteins are disclosed. Glycosylation and the KEX2protease activity are only important, in this context, upon expressionin yeast.

WO 88/06161 mentions various mutants which theoretically may beconformationally and antigenically neutral. The only actually performedmutations are Met²->Ile² and Ile¹³¹->Leu¹³¹. It is not disclosed whetherthe contemplated neutralities were obtained for these two mutations.

WO 91/00350 discloses nonglycosylated hIL-3 analog proteins, forexample, hIL-3 (Pro⁸Asp¹⁵Asp⁷⁰), Met³ rhul-3 (Pro⁸Asp¹⁵Asp⁷⁰); Thr⁴rhuL-3 (Pro⁸Asp¹⁵Asp⁷⁰) and Thr⁶ rhuIL-3 (Pro⁸Asp¹⁵Asp⁷⁰). It is saidthat these protein compositions do not exhibit certain adverse sideeffects associated with native hIL-3 such as urticaria resulting frominfiltration of mast cells and lymphocytes into the dermis. Thedisclosed analog hIL-3 proteins may have N termini at Met³, Thr⁴, orThr⁶.

WO 90/12874 discloses cysteine added variants (CAVs) of IL-3 which haveat least one Cys residue substituted for a naturally occurring aminoacid residue.

U.S. Pat. No. 4,810,643 discloses the DNA sequence encoding human G-CSF.

WO 91/02754 discloses a fusion protein composed of GM-CSF and IL-3 whichhas increased biological activity compared to GM-CSF or IL-3 alone. Alsodisclosed are nonglycosylated IL-3 and GM-CSF analog proteins ascomponents of the fusion.

WO 92/04455 discloses fusion proteins composed of IL-3 fused to alymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9,IL-11, EPO and G-CSF.

SUMMARY OF THE INVENTION

The present invention encompasses recombinant human interleukin-3(hIL-3) variant or mutant proteins (muteins) fused to a second colonystimulating factor (CSF), cytokine, lymphokine, interleukin,hematopoietic growth factor (herein collectively referred to as “colonystimulating factors”) or IL-3 variant with or without a linker. ThesehIL-3 muteins contain amino acid substitutions and may also have aminoacid deletions at either/or both the N- and C-termini. This inventionencompasses mixed function colony stimulating factors formed fromcovalently linked polypeptides, each of which may act through adifferent and specific cell receptor to initiate complementarybiological activities.

Novel compounds of this invention are represented by the formulasR₁-L-R₂, R₂-L-R₁, R₁-R₂ or R₂-R₁where R₁ is a hIL-3 variant which contains one to three amino acidsubstitutions and which may have portions of the hIL-3 molecule deleted,R₂ is a CSF with a different but complementary activity. The R₁polypeptide is fused either directly or through a linker segment to theR₂ polypeptide. Thus L represents a chemical bound or polypeptidesegment to which both R₁ and R₂ are fused. Preferably, these mutant IL-3polypeptides of the present invention contain one to three amino acidswhich differ from the amino acids found at the corresponding positionsin the native hIL-3 polypeptide. The invention also relates topharmaceutical compositions containing the fusion molecules, DNA codingfor the fusion molecules, and methods for using the fusion molecules.Additionally, the present invention relates to recombinant expressionvectors comprising nucleotide sequences encoding the hIL-3 fusionmolecules, related microbial expression systems, and processes formaking the fusion molecules using the microbial expression systems.

These fusion molecules may be characterized by having the usual activityof both of the peptides forming the fusion molecule or it may be furthercharacterized by having a biological or physiological activity greaterthan simply the additive function of the presence of IL-3 or the secondcolony stimulating factor alone. The fusion molecule may alsounexpectedly provide an enhanced effect on the activity or an activitydifferent from that expected by the presence of IL-3 or the secondcolony stimulating factor or IL-3 variant. The fusion molecule may alsohave an improved activity profile which may include reduction ofundesirable biological activities associated with native hIL-3.

The present invention also includes mutants of hIL-3 in which from 1 to14 amino acids have been deleted from the N-terminus and/or from 1 to 15amino acids have been deleted from the C-terminus, containing one tothree amino acid substitutions, to which a second colony stimulatingfactor or IL-3 variant has been fused. Preferred fusion molecules of thepresent invention are composed of hIL-3 variants in which amino acids 1to 14 have been deleted from the N-terminus, amino acids 126 to 133 havebeen deleted from the C-terminus, and contains from about one to threeamino acid substitutions in the polypeptide sequence fused to secondcolony stimulating factor or IL-3 variant.

The present invention also provides fusion molecules which may functionas IL-3 antagonists or as discrete antigenic fragments for theproduction of antibodies useful in immunoassay and immunotherapyprotocols. Antagonists of hIL-3 would be particularly useful in blockingthe growth of certain cancer cells like AML, CML and certain types of Blymphoid cancers. Other conditions where antagonists would be usefulinclude those in which certain blood cells are produced at abnormallyhigh numbers or are being activated by endogenous ligands. Antagonistswould effectively compete for ligands, presumably naturally occurringhemopoietins including and not limited to IL-3, GM-CSF and IL-5, whichmight trigger or augment the growth of cancer cells by virtue of theirability to bind to the IL-3 receptor complex while intrinsic activationproperties of the ligand are diminished. IL-3, GM-CSF and/or IL-5 alsoplay a role in certain asthmatic responses. An antagonist of the IL-3receptor may have the utility in this disease by blockingreceptor-mediated activation and recruitment of inflammatory cells.

In addition to the use of the fusion molecules of the present inventionin vivo, it is envisioned that in vitro uses would include the abilityto stimulate bone marrow and blood cell activation and growth beforeinfusion into patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the human IL-3 gene for E. coli expression (pMON5873),encoding the polypeptide sequence of natural (wild type) human IL-3 [SEQID NO:9], plus an initiator methionine, as expressed in E. coli, withthe amino acids numbered from the N-terminus of the natural hIL-3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses recombinant human interleukin-3(hIL-3) variant or mutant proteins (muteins) fused to a second colonystimulating factor (CSF), cytokine, lymphokine, interleukin,hematopoietic growth factor or IL-3 variant with or without a linker.This invention encompasses mixed function colony stimulating factorsformed from covalently linked polypeptides, each of which may actthrough a different and specific cell receptor to initiate complementarybiological activities. Hematopoiesis requires a complex series ofcellular events in which stem cells generate continuously into largepopulations of maturing cells in all major lineages. There are currentlyat least 20 known regulators with hematopoietic proliferative activity.Most of these proliferative regulators can stimulate one or another typeof colony formation in vitro, the precise pattern of colony formationstimulated by each regulator is quite distinctive. No two regulatorsstimulate exactly the same pattern of colony formation, as evaluated bycolony numbers or, more importantly, by the lineage and maturationpattern of the cells making up the developing colonies. Proliferativeresponses can most readily be analyzed in simplified in vitro culturesystems. Three quite different parameters can be distinguished:alteration in colony size, alteration in colony numbers and celllineage. Two or more factors may act on the progenitor cell, inducingthe formation of larger number of progeny thereby increasing the colonysize. Two or more factors may allow increased number of progenitor cellsto proliferate either because distinct subsets of progenitors cellsexist that respond exclusively to one factor or because some progenitorsrequire stimulation by two or more factors before being able to respond.Activation of additional receptors on a cell by the use of two or morefactors is likely to enhance the mitotic signal because of coalescenceof initially differing signal pathways into a common final pathwayreaching the nucleus (Metcalf, 1989). Other mechanisms could explainsynergy. For example, if one signalling pathway is limited by anintermediate activation of an additional signalling pathway by a secondfactor may result in a superadditive response. In some cases, activationof one receptor type can induce a enhanced expression of other receptors(Metcalf, 1993). Two or more factors may result in a different patternof cell lineages then from a single factor. The use of fusion moleculesmay have the potential clinical advantage resulting from a proliferativeresponse that is not possible by any single factor.

Hematopoietic and other growth factors can be grouped in to two distinctfamilies of related receptors: (1) tyrosine kinase receptors, includingthose for epidermal growth factor, M-CSF (Sherr, 1990) and SCF (Yardenet al., 1987): and (2) hematopoietic receptors, not containing atyrosine kinase domain, but exhibiting obvious homology in theirextracellular domain (Bazan, 1990). Included in this later group areerythropoietin (EPO) (D'Andrea et al., 1989), GM-CSF (Gearing et al.,1989), IL-3 (Kitamura et al., 1991), G-CSF (Fukunaga et al., 1990), IL-4(Harada et al., 1990), IL-5 ((Takaki et al., 1990), IL-6 (Yamasaki etal., 1988), IL-7 (Goodwin et al., 1990), LIF (Gearing et al., 1991) andIL-2 (Cosman et al., 1987). Most of the later group of receptors existsin high-affinity form as a heterodimers. After ligand binding, thespecific α-chains become associated with at least one other receptorchain (β-chain, γ-chain). Many of these factors share a common receptorsubunit. The α-chains for GM-CSF, IL-3 and IL-5 share the same β-chain(Miyajima et al., 1992) and receptor complexes for IL-6, LIF and IL-11share a common β-chain (gp130) (Taga et al., 1989; Taga et al., 1992;Gearing et al., 1992). The receptor complexes of IL-2, IL-4 and IL-7share a common γ-chain (Motonari et al., 1993; Russell et al., 1993;Masayuki et al., 1993).

The use of multiple factors may also have potential advantage bylowering the demands placed on factor-producing cells and theirinduction systems. If there are limitations in the ability of a cell toproduce a factor then by lowering the required concentrations of each ofthe factors by using them in combination may usefully reduce demands onthe factor-producing cells. The use of multiple factors may lower theamount of the factors that would be needed, probably reducing thelikelihood of adverse responses.

Novel compounds of this invention are represented by a formula selectedfrom the group consisting ofR₁-L-R₂, R₂-L-R₁, R₁-R₂ or R₂-R₁where R₁ is a hIL-3 variant which contains one to three amino acidsubstitutions and which may have portions of the hIL-3 molecule deletedas is disclosed in co-pending U.S. Patent Application Ser. No.PCT/US93/11197, R2 is a colony stimulating factor with a different butcomplementary activity. By complementary activity is meant activitywhich enhances or changes the response to another cell modulator. The R1polypeptide is fused either directly or through a linker segment to theR2 polypeptide. The term “directly” defines fusions in which thepolypeptides are joined without a peptide linker. Thus L represents achemical bound or polypeptide segment to which both R1 and R2 are fusedin frame, most commonly L is a linear peptide to which R1 and R2 arebound by amide bonds linking the carboxy terminus of R1 to the aminoterminus of L and carboxy terminus of L to the amino terminus of R2. By“fused in frame” is meant that there is no translation termination ordisruption between the reading frames of R1 and R2. A non-exclusive listof other growth hormone, colony stimulating factors (CSFs), cytokine,lymphokine, interleukin, hematopoietic growth factor within thedefinition of R2, which can be fused to a hIL-3 variant of the presentinvention include GM-CSF, CSF-1, G-CSF, Meg-CSF, M-CSF, erythropoietin(EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, LIF, flt3 ligand, human growth hormone, B-cell growthfactor, B-cell differentiation factor, eosinophil differentiation factorand stem cell factor (SCF) also known as steel factor or c-kit ligand.Additionally, this invention encompasses the use of modified R2molecules or mutated or modified DNA sequences encoding these R2molecules. The present invention also includes fusion molecules in whichR2 is a hIL-3 variant which contains one to three amino acidsubstitutions and which may have portions of the hIL-3 molecule deleted.

The linking group (L) is generally a polypeptide of between 1 and 500amino acids in length. The linkers joining the two molecules arepreferably designed to (1) allow the two molecules to fold and actindependently of each other, (2) not have a propensity for developing anordered secondary structure which could interfere with the functionaldomains of the two proteins, (3) have minimal hydrophobic or chargedcharacteristic which could interact with the functional protein domainsand (4) provide steric separation of R1 and R2 such that R1 and R2 couldinteract simultaneously with their corresponding receptors on a singlecell. Typically surface amino acids in flexible protein regions includeGly, Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

Preferred linkers of the present invention include sequences selectedfrom the group of formulas:(Gly₃Ser)_(n), (Gly₄Ser)_(n), (Gly₅Ser)_(n), (Gly_(n)Ser)_(n) or(AlaGlySer)_(n)

One example of a highly-flexible linker is the (GlySer)-rich spacerregion present within the pIII protein of the filamentousbacteriophages, e.g. bacteriophages M13 or fd (Schaller et al., 1975).This region provides a long, flexible spacer region between two domainsof the pIII surface protein. The spacer region consists of the aminoacid sequence:

GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySer GlyGlyGlySer [Seq. Id.No. 11]

The present invention also includes linkers in which an endopeptidaserecognition sequence is included. Such a cleavage site may be valuableto separate the individual components of the fusion to determine if theyare properly folded and active in vitro. Examples of variousendopeptidases include, but are not limited to, Plasmin, Enterokinase,Kallikerin, Urokinase, Tissue Plasminogen activator, clostripain,Chymosin, Collagenase, Russell's Viper Venom Protease, Postprolinecleavage enzyme, V8 protease, Thrombin and factor Xa.

Peptide linker segments from the hinge region of heavy chainimmunoglobulins IgG, IgA, IgM, IgD or IgE provide an angularrelationship between the attached polypeptides. Especially useful arethose hinge regions where the cysteines are replaced with serines.Preferred linkers of the present invention include sequences derivedfrom murine IgG gamma 2b hinge region in which the cysteins have beenchanged to serines. These linkers may also include an endopeptidasecleavage site. Examples of such linkers include the following sequencesselected from the group of sequences

IleSerGluProSerGlyProIleSerThrIleAsnProSerProProSerLysGluSerHisLysSerPro [Seq. Id. No. 12]

IleGluGlyArgIleSerGluProSerGlyProIleSerThrIleAsnProSerProProSerLysGluSerHisLysSerPro [Seq. Id. No. 11]

The present invention is, however, not limited by the form, size ornumber of linker sequences employed and the only requirement of thelinker is that functionally it does not interfere adversely with thefolding and function of the individual molecules of the fusion.

An alternative method for connecting two hematopoietic growth factors isby means of a non-covalent interaction. Such complexed proteins can bedescribed by one the formulae:R1-C1+R2-C2; or C1-R1+C2-R2; C1-R1+R2-C2; or C1-R1+R2-C2.where R1 is a hIL-3 variant which contains one to three amino acidsubstitutions and which may have portions of the hIL-3 molecule deleted,R2 is a colony stimulating factor with a different but complementaryactivity. A non-exclusive list of other colony stimulating factors(CSFs), cytokine, lymphokine, interleukin, hematopoietic growth factorwithin the definition of R2, which can be fused to a hIL-3 variant ofthe present invention include GM-CSF, CSF-1, G-CSF, Meg-CSF, M-CSF,erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, LIF, B-cell growth factor, B-celldifferentiation factor, eosinophil differentiation factor and stem cellfactor (SCF) also known as steel factor or c-kit ligand. Domains C1 andC2 are either identical or non-identical chemical structures, typicallyproteinaceous, which can form a non-covalent, specific association.Complexes between C1 and C2 result in a one-to-one stoichiometricrelationship between R1 and R2 for each complex. Examples of domainswhich associate are “leucine zipper” domains of transcription factors,dimerization domains of bacterial transcription repressors andimmunoglobulin constant domains. Covalent bonds link R1 and C1, and R2and C2, respectively. As indicated in the formulae, the domains C1 andC2 can be present either at the N-terminus or C-terminus of theircorresponding hematopoietic growth factor (R). These multimerizationdomains (C1 and C2) include those derived from the bZIP family ofproteins (Abel et al., 1989; Landshulz et al., 1988; Pu et al., 1993;Korarides et al., 1988) as well as multimerization domains of thehelix-loop-helix family of proteins (Abel et al., 1989; Murre et al.,1989; Tapscott et al., 1988; Fisher et al., 1991). Preferred fusions ofthe present invention include colony stimulating factors dimerized byvirtue of their incorporation as translational fusions the leucinezipper dimerization domains of the bZIP family proteins Fos and Jun. Theleucine zipper domain of Jun is capable of interacting with identicaldomains. On the other hand, the leucine zipper domain of Fos interactswith the Jun leucine zipper domain, but does not interact with other Fosleucine zipper domains. Mixtures of Fos and Jun predominantly result information of Fos-Jun heterodimers. Consequently, when fused to colonystimulating factors, the Jun domain can be used to direct the formationof either homo or heterodimers. Preferential formation of heterodimerscan be achieved if one of the colony stimulating factor partner isengineered to possess the Jun leucine zipper domain while the other isengineered to possess the Fos zipper.

Peptides may also be added to facilitate purification or identificationof fusion proteins (e.g., poly-His). A highly antigenic peptide may alsobe added that would enable rapid assay and facile purification of thefusion protein by a specific monoclonal antibody.

The present invention relates to novel fusion molecules composed ofnovel variants of human interleukin-3 (hIL-3) in which amino acidsubstitutions have been made at one to three positions in amino acidsequence of the polypeptide fused to second colony stimulating factor orIL-3 variant. Preferred fusion molecules of the present invention are(15–125)hIL-3 deletion mutants which have deletions of amino acids 1 to14 at the N-terminus and 126 to 133 at the C-terminus and which alsohave one to three amino acid substitutions in the polypeptide fused tosecond colony stimulating factor or IL-3 variant. The present inventionincludes mutant polypeptides comprising minimally amino acids residues15 to 118 of hIL-3 with or without additional amino acid extensions tothe N-terminus and/or C-terminus which further contain one to threeamino acid substitutions in the amino acid sequence of the polypeptidefused to another colony stimulating factor or IL-3 variant.

As used herein human interleukin-3 corresponds to the amino acidsequence (1–133) as depicted in FIG. 1 and (15–125) hIL-3 corresponds tothe 15 to 125 amino acid sequence of the hIL-3 polypeptide. Naturallyoccurring variants of hIL-3 polypeptide amino acids are also included inthe present invention (for example, the allele in which proline ratherthan serine is at position 8 in the hIL-3 polypeptide sequence) as arevariant hIL-3 molecules which are modified post-translationally (e.g.glycosylation).

“Mutant amino acid sequence,” “mutant protein” or “mutant polypeptide”refers to a polypeptide having an amino acid sequence which varies froma native sequence or is encoded by a nucleotide sequence intentionallymade variant from a native sequence. “Mutant protein,” “variant protein”or “mutein” means a protein comprising a mutant amino acid sequence andincludes polypeptides which differ from the amino acid sequence ofnative hIL-3 due to amino acid deletions, substitutions, or both.“Native sequence” refers to an amino acid or nucleic acid sequence whichis identical to a wild-type or native form of a gene or protein.

Human IL-3 can be characterized by its ability to stimulate colonyformation by human hematopoietic progenitor cells. The colonies formedinclude erythroid, granulocyte, megakaryocyte, granulocytic macrophagesand mixtures thereof. Human IL-3 has demonstrated an ability to restorebone marrow function and peripheral blood cell populations totherapeutically beneficial levels in studies performed initially inprimates and subsequently in humans (Gillio, A. P., et al. (1990);Ganser, A, et al. (1990); Falk, S., et al. (1991). Additional activitiesof hIL-3 include the ability to stimulate leukocyte migration andchemotaxis; the ability to prime human leukocytes to produce high levelsof inflammatory mediators like leukotrienes and histamine; the abilityto induce cell surface expression of molecules needed for leukocyteadhesion; and the ability to trigger dermal inflammatory responses andfever. Many or all of these biological activities of hIL-3 involvesignal transduction and high affinity receptor binding. Fusion moleculesof the present invention may exhibit useful properties such as havingsimilar or greater biological activity when compared to native hIL-3 orby having improved half-life or decreased adverse side effects, or acombination of these properties. They may also be useful as antagonists.Fusion molecules which have little or no activity when compared tonative hIL-3 may still be useful as antagonists, as antigens for theproduction of antibodies for use in immunology or immunotherapy, asgenetic probes or as intermediates used to construct other useful hIL-3muteins.

The novel fusion molecules of the present invention will preferably haveat least one biological property of human IL-3 and the other colonystimulating factor or IL-3 variant to which it is fused and may havemore than one IL-3-like biological property, or an improved property, ora reduction in an undesirable biological property of human IL-3. Somemutant polypeptides of the present invention may also exhibit animproved side effect profile. For example, they may exhibit a decreasein leukotriene release or histamine release when compared to nativehIL-3 or (15–125) hIL-3. Such hIL-3 or hIL-3-like biological propertiesmay include one or more of the following biological characteristics andin vivo and in vitro activities.

One such property is the support of the growth and differentiation ofprogenitor cells committed to erythroid, lymphoid, and myeloid lineages.For example, in a standard human bone marrow assay, an IL-3-likebiological property is the stimulation of granulocytic type colonies,megakaryocytic type colonies, monocyte/macrophage type colonies, anderythroid bursts. Other IL-3-like properties are the interaction withearly multipotential stem cells, the sustaining of the growth ofpluripotent precursor cells, the ability to stimulate chronicmyelogenous leukemia (CML) cell proliferation, the stimulation ofproliferation of mast cells, the ability to support the growth ofvarious factor-dependent cell lines, and the ability to trigger immaturebone marrow cell progenitors. Other biological properties of IL-3 havebeen disclosed in the art. Human IL-3 also has some biologicalactivities which may in some cases be undesirable, for example theability to stimulate leukotriene release and the ability to stimulateincreased histamine synthesis in spleen and bone marrow cultures and invivo.

Biological activity of hIL-3 and hIL-3 fusion proteins of the presentinvention is determined by DNA synthesis by human acute myelogenousleukemia cells (AML). The factor-dependent cell line AML 193 was adaptedfor use in testing biological activity. The biological activity of hIL-3and hIL-3 fusion proteins of the present invention is also determined bycounting the colony forming units in a bone marrow assay.

Other in vitro cell based assays may also be useful to determine theactivity of the fusion molecules depending on the colony stimulatingfactors that comprise the fusion. The following are examples of otheruseful assays.

TF-1 proliferation assay: The TF-1 cell line was derived from bonemarrow of a patient with erythroleukemia (Kitamura et al., 1989). TF-1cells respond to IL-3, GM-CSF, EPO and IL-5.

32D proliferation assay: 32D is a murine IL-3 dependent cell line whichdoes not respond to human IL-3 but does respond to human G-CSF which isnot species restricted.

T1165 proliferation assay: T1165 cells are a IL-6 dependent murine cellline (Nordan et al., 1986) which respond to IL-6 and IL-11.

Human Plasma Clot meg-CSF Assay: Used to assay megakaryocyte colonyformation activity (Mazur et al., 1981).

One object of the present invention is to provide hIL-3 variant with oneto three amino acid substitutions in the polypeptide sequence fused to asecond colony stimulating factor or IL-3 variant, which have similar orimproved biological activity in relation to native hIL-3 or the secondcolony stimulating factor or IL-3 variant.

The hIL-3 variant fusion molecules of the present invention may havehIL-3 or hIL-3-like activity. For example, they may possess one or moreof the biological activities of native hIL-3 and may be useful instimulating the production of hematopoietic cells by human or primateprogenitor cells. The fusion molecules of the present invention andpharmaceutical compositions containing them may be useful in thetreatment of conditions in which hematopoietic cell populations havebeen reduced or destroyed due to disease or to treatments such asradiation and/or chemotherapy. Pharmaceutical compositions containingfusion molecules of the present invention can be administeredparenterally, intravenously, or subcutaneously.

Native hIL-3 possesses considerable inflammatory activity and has beenshown to stimulate synthesis of the arachidonic acid metabolites LTC₄,LTD₄, and LTE₄; histamine synthesis and histamine release. Humanclinical trials with native hIL-3 have documented inflammatory responses(Biesma, et al., BLOOD, 80:1141–1148 (1992) and Postmus, et al., J.CLIN. ONCOL., 10:1131–1140 (1992)). A recent study indicates thatleukotrienes are involved in IL-3 actions in vivo and may contributesignificantly to the biological effects of IL-3 treatment (Denzlinger,C., et al., BLOOD, 81:2466–2470 (1993))

Some fusion molecules of the present invention may have an improvedtherapeutic profile as compared to native hIL-3. For example, somefusion molecules of the present invention may have a similar or morepotent growth factor activity relative to native hIL-3 without having asimilar or corresponding increase in the stimulation of leukotriene orhistamine. These fusion molecules would be expected to have a morefavorable therapeutic profile since the amount of polypeptide whichneeds to be given to achieve the desired growth factor activity (e.g.cell proliferation) would have a lesser leukotriene or histaminestimulating effect. In studies with native hIL-3, the stimulation ofinflammatory factors has been an undesirable side effect of thetreatment. Reduction or elimination of the stimulation of mediators ofinflammation would provide an advantage over the use of native hIL-3.

Novel fusion molecules of the present invention may also be useful asantagonists which block the hIL-3 receptor by binding specifically to itand preventing binding of the agonist.

One potential advantage of the novel fusion molecules of the presentinvention, particularly those which retain activity similar to or betterthan that of native hIL-3, is that it may be possible to use a smalleramount of the biologically active mutein to produce the desiredtherapeutic effect. This may make it possible to reduce the number oftreatments necessary to produce the desired therapeutic effect. The useof smaller amounts may also reduce the possibility of any potentialantigenic effects or other possible undesirable side effects. Forexample, if a desired therapeutic effect can be achieved with a smalleramount of polypeptide it may be possible to reduce or eliminate sideeffects associated with the administration of native IL-3 such as thestimulation of leukotriene and/or histamine release. The novel fusionmolecules of the present invention may also be useful in the activationof stem cells or progenitors which have low receptor numbers.

The present invention also includes the DNA sequences which code for thefusion proteins, DNA sequences which are substantially similar andperform substantially the same function, and DNA sequences which differfrom the DNAs encoding the fusion molecules of the invention only due tothe degeneracy of the genetic code. Also included in the presentinvention are; the oligonucleotide intermediates used to construct themutant DNAs; and the polypeptides coded for by these oligonucleotides.These polypeptides may be useful as antagonists or as antigenicfragments for the production of antibodies useful in immunoassay andimmunotherapy protocols.

Compounds of this invention are preferably made by genetic engineeringtechniques now standard in the art U.S. Pat. No. 4,935,233 and Sambrooket al., “Molecular Cloning. A Laboratory Manual”, Cold Spring HarborLaboratory (1989)]. One method of creating the preferred hIL-3 (15–125)mutant genes is cassette mutagenesis [Wells, et al. (1985)] in which aportion of the coding sequence of hIL-3 in a plasmid is replaced withsynthetic oligonucleotides that encode the desired amino acidsubstitutions in a portion of the gene between two restriction sites. Ina similar manner amino acid substitutions could be made in thefull-length hIL-3 gene, or genes encoding variants of hIL-3 in whichfrom 1 to 14 amino acids have been deleted from the N-terminus and/orfrom 1 to 15 amino acids have been deleted from the C-terminus. Whenproperly assembled these oligonucleotides would encode hIL-3 variantswith the desired amino acid substitutions and/or deletions from theN-terminus and/or C-terminus. These and other mutations could be createdby those skilled in the art by other mutagenesis methods including;oligonucleotide-directed mutagenesis [Zoller and Smith (1982, 1983,1984), Smith (1985), Kunkel (1985), Taylor, et al. (1985), Deng andNickoloff (1992)] or polymerase chain reaction (PCR) techniques [Saiki,(1985)].

Pairs of complementary synthetic oligonucleotides encoding the desiredgene can be made and annealed to each other. The DNA sequence of theoligonucleotide would encode sequence for amino acids of desired genewith the exception of those substituted and/or deleted from thesequence.

Plasmid DNA can be treated with the chosen restriction endonucleasesthen ligated to the annealed oligonucleotides. The ligated mixtures canbe used to transform competent JM101 cells to resistance to anappropriate antibiotic. Single colonies can be picked and the plasmidDNA examined by restriction analysis and/or DNA sequencing to identifyplasmids with the desired genes.

Fusing of the DNA sequences of the hIL-3 variant with the DNA sequenceof the other colony stimulating factor or IL-3 variant may beaccomplished by the use of intermediate vectors. Alternatively one genecan be cloned directly into a vector containing the other gene. Linkersand adapters can be used for joining the DNA sequences, as well asreplacing lost sequences, where a restriction site was internal to theregion of interest. Thus genetic material (DNA) encoding onepolypeptide, peptide linker, and the other polypeptide is inserted intoa suitable expression vector which is used to transform bacteria, yeast,insect cell or mammalian cells. The transformed organism is grown andthe protein isolated by standard techniques. The resulting product istherefore a new protein which has a hIL-3 variant joined by a linkerregion to a second colony stimulating factor or IL-3 variant.

Another aspect of the present invention provides plasmid DNA vectors foruse in the expression of these novel fusion molecules. These vectorscontain the novel DNA sequences described above which code for the novelpolypeptides of the invention. Appropriate vectors which can transformmicroorganisms capable of expressing the fusion molecules includeexpression vectors comprising nucleotide sequences coding for the fusionmolecules joined to transcriptional and translational regulatorysequences which are selected according to the host cells used.

Vectors incorporating modified sequences as described above are includedin the present invention and are useful in the production of the fusionpolypeptides. The vector employed in the method also contains selectedregulatory sequences in operative association with the DNA codingsequences of the invention and capable of directing the replication andexpression thereof in selected host cells.

As another aspect of the present invention, there is provided a methodfor producing the novel fusion molecules. The method of the presentinvention involves culturing a suitable cell or cell line, which hasbeen transformed with a vector containing a DNA sequence coding forexpression of a novel hIL-3 variant fusion molecule. Suitable cells orcell lines may be bacterial cells. For example, the various strains ofE. coli are well-known as host cells in the field of biotechnology.Examples of such strains include E. coli strains JM101 [Yanish-Perron,et al. (1985)] and MON105 [Obukowicz, et al. (1992)]. Also included inthe present invention is the expression of the fusion protein utilizinga chromosomal expression vector for E. coli based on the bacteriophageMu (Weinberg et al., 1993). Various strains of B. subtilis may also beemployed in this method. Many strains of yeast cells known to thoseskilled in the art are also available as host cells for expression ofthe polypeptides of the present invention. When expressed in the E. colicytoplasm, the above-mentioned mutant hIL-3 variant fusion molecules ofthe present invention may also be constructed with Met-Ala- at theN-terminus so that upon expression the Met is cleaved off leaving Ala atthe N-terminus. The fusion molecules of the present invention mayinclude fusion polypeptides having Met-, Ala- or Met-Ala- attached tothe N-terminus. When the fusion molecules are expressed in the cytoplasmof E. coli, polypeptides with and without Met attached to the N-terminusare obtained. The N-termini of proteins made in the cytoplasm of E. coliare affected by posttranslational processing by methionineaminopeptidase (Ben-Bassat et al., 1987) and possibly by otherpeptidases. These mutant fusion molecules may also be expressed in E.coli by fusing a signal peptide to the N-terminus. This signal peptideis cleaved from the polypeptide as part of the secretion process.Secretion in E. coli can be used to obtain the correct amino acid at theN-terminus (e.g., Asn¹⁵ in the (15–125) hIL-3 polypeptide) due to theprecise nature of the signal peptidase. This is in contrast to theheterogeneity which may be observed at the N-terminus of proteinsexpressed in the cytoplasm in E. coli.

Also suitable for use in the present invention are mammalian cells, suchas Chinese hamster ovary cells (CHO). General methods for expression offoreign genes in mammalian cells are reviewed in: Kaufman, R. J. (1987)High level production of proteins in mammalian cells, in GeneticEngineering, Principles and Methods, Vol. 9, J. K. Setlow, editor,Plenum Press, New York. An expression vector is constructed in which astrong promoter capable of functioning in mammalian cells drivestranscription of a eukaryotic secretion signal peptide coding region,which is translationally fused to the coding region for the fusionmolecule. For example, plasmids such as pcDNA I/Neo, pRc/RSV, andpRc/CMV (obtained from Invitrogen Corp., San Diego, Calif.) can be used.The eukaryotic secretion signal peptide coding region can be from thehIL-3 gene itself or it can be from another secreted mammalian protein(Bayne, M. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84, 2638–2642).After construction of the vector containing the hIL-3 variant gene, thevector DNA is transfected into mammalian cells. Such cells can be, forexample, the COS7, HeLa, BHK, CHO, or mouse L lines. The cells can becultured, for example, in DMEM media (JRH Scientific). The hIL-3 variantsecreted into the media can be recovered by standard biochemicalapproaches following transient expression 24–72 hours after transfectionof the cells or after establishment of stable cell lines followingselection for neomycin resistance. The selection of suitable mammalianhost cells and methods for transformation, culture, amplification,screening and product production and purification are known in the art.See, e.g., Gething and Sambrook, Nature, 293:620–625 (1981), oralternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750–1759 (1985) orHowley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cellline is the monkey COS-1 cell line. A similarly useful mammalian cellline is the CV-1 cell line.

Where desired, insect cells may be utilized as host cells in the methodof the present invention. See, e.g. Miller et al, Genetic Engineering,8:277–298 (Plenum Press 1986) and references cited therein. In addition,general methods for expression of foreign genes in insect cells usingBaculovirus vectors are described in: Summers, M. D. and Smith, G. E.(1987)—A manual of methods for Baculovirus vectors and insect cellculture procedures, Texas Agricultural Experiment Station Bulletin No.1555. An expression vector is constructed comprising a Baculovirustransfer vector, in which a strong Baculovirus promoter (such as thepolyhedron promoter) drives transcription of a eukaryotic secretionsignal peptide coding region, which is translationally fused to thecoding region for the fusion polypeptide. For example, the plasmidpVL1392 (obtained from Invitrogen Corp., San Diego, Calif.) can be used.After construction of the vector carrying the gene encoding the fusionpolypeptide, two micrograms of this DNA is cotransfected with onemicrogram of Baculovirus DNA (see Summers & Smith, 1987) into insectcells, strain SF9. Pure recombinant Baculovirus carrying the fusionmolecule is used to infect cells cultured, for example, in Excell 401serum-free medium (JRH Biosciences, Lenexa, Kans.). The fusion moleculesecreted into the medium can be recovered by standard biochemicalapproaches. Supernatants from mammalian or insect cells expressing thefusion protein can be first concentrated using any of an number ofcommercial concentration units.

The fusion molecules of the present invention may be useful in thetreatment of diseases characterized by a decreased levels of eithermyeloid, erythroid, lymphoid, or megakaryocyte cells of thehematopoietic system or combinations thereof. In addition, they may beused to activate mature myeloid and/or lymphoid cells. Among conditionssusceptible to treatment with the polypeptides of the present inventionis leukopenia, a reduction in the number of circulating leukocytes(white cells) in the peripheral blood. Leukopenia may be induced byexposure to certain viruses or to radiation. It is often a side effectof various forms of cancer therapy, e.g., exposure to chemotherapeuticdrugs, radiation and of infection or hemorrhage. Therapeutic treatmentof leukopenia with these fusion molecules of the present invention mayavoid undesirable side effects caused by treatment with presentlyavailable drugs.

The fusion molecules of the present invention may be useful in thetreatment of neutropenia and, for example, in the treatment of suchconditions as aplastic anemia, cyclic neutropenia, idiopathicneutropenia, Chediak-Higashi syndrome, systemic lupus erythematosus(SLE), leukemia, myelodysplastic syndrome and myelofibrosis.

The fusion molecule of the present invention may be useful in thetreatment or prevention of thrombocytopenia. Currently the only therapyfor thrombocytopenia is platelet transfusions which are costly and carrythe significant risks of infection (HIV, HBV) and alloimunization. Thefusion molecule may alleviate or diminish the need for platelettransfusions. Severe thrombocytopenia may result from genetic defectssuch as Fanconi's Anemia, Wiscott-Aldrich, or May-Hegglin syndromes.Acquired thrombocytopenia may result from auto- or allo-antibodies as inImmune Thrombocytopenia Purpura, Systemic Lupus Erythromatosis,hemolytic anemia, or fetal maternal incompatibility. In addition,splenomegaly, disseminated intravascular coagulation, thromboticthrombocytopenic purpura, infection or prosthetic heart valves mayresult in thrombocytopenia. Severe thrombocytopenia may also result fromchemotherapy and/or radiation therapy or cancer. Thrombocytopenia mayalso result from marrow invasion by carcinoma, lymphoma, leukemia orfibrosis.

The fusion molecules of the present invention may be useful in themobilization of hematopoietic progenitors and stem cells into peripheralblood. Peripheral blood derived progenitors have been shown to beeffective in reconstituting patients in the setting of autologous marrowtransplantation. Hematopoietic growth factors including G-CSF and GM-CSFhave been shown to enhance the number of circulating progenitors andstem cells in the peripheral blood. This has simplified the procedurefor peripheral stem cell collection and dramatically decreased the costof the procedure by decreasing the number of pheresis required. Thefusion molecule may be useful in mobilization of stem cells and furtherenhance the efficacy of peripheral stem cell transplantation.

Another projected clinical use of growth factors has been in the invitro activation of hematopoietic progenitors and stem cells for genetherapy. In order to have the gene of interest incorporated into thegenome of the hematopoietic progenitor or stem cell one needs tostimulate cell division and DNA replication. Hematopoietic stem cellscycle at a very low frequency which means that growth factors may beuseful to promote gene transduction and thereby enhance the clinicalprospects for gene therapy.

Many drugs may cause bone marrow suppression or hematopoieticdeficiencies. Examples of such drugs are AZT, DDI, alkylating agents andanti-metabolites used in chemotherapy, antibiotics such aschloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs,phenothiazones, tranquilizers such as meprobamate, analgesics such asaminopyrine and dipyrone, anti-convulsants such as pheytoin orcarbamazepine, and antithyroids such as propylthiouracil andmethimazole, and diuretics. The fusion molecules of the presentinvention may be useful in preventing or treating the bone marrowsuppression or hematopoietic deficiencies which often occur in patientstreated with these drugs.

Hematopoietic deficiencies may also occur as a result of viral,microbial or parasitic infections and as a result of treatment for renaldisease or renal failure, e.g., dialysis. The fusion molecules of thepresent invention may be useful in treating such hematopoieticdeficiency.

The treatment of hematopoietic deficiency may include administration ofa pharmaceutical composition containing the fusion molecules to apatient. The fusion molecules of the present invention may also beuseful for the activation and amplification of hematopoietic precursorcells by treating these cells in vitro with the fusion proteins of thepresent invention prior to injecting the cells into a patient.

Various immunodeficiencies e.g., in T and/or B lymphocytes, or immunedisorders, e.g., rheumatoid arthritis, may also be beneficially affectedby treatment with the fusion molecules of the present invention.Immunodeficiencies may be the result of viral infections e.g. HTLVI,HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or theresult of other medical treatment. The fusion molecules of the presentinvention may also be employed, alone or in combination with otherhematopoietins, in the treatment of other blood cell deficiencies,including thrombocytopenia (platelet deficiency), or anemia. Other usesfor these novel polypeptides are in the treatment of patients recoveringfrom bone marrow transplants in vivo and ex vivo, and in the developmentof monoclonal and polyclonal antibodies generated by standard methodsfor diagnostic or therapeutic use.

Other aspects of the present invention are methods and therapeuticcompositions for treating the conditions referred to above. Suchcompositions comprise a therapeutically effective amount of one or moreof the fusion molecules of the present invention in a mixture with apharmaceutically acceptable carrier. This composition can beadministered either parenterally, intravenously or subcutaneously. Whenadministered, the therapeutic composition for use in this invention ispreferably in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such a parenterally acceptableprotein solution, having due regard to pH, isotonicity, stability andthe like, is within the skill of the art.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician consideringvarious factors which modify the action of drugs, e.g. the condition,body weight, sex and diet of the patient, the severity of any infection,time of administration and other clinical factors. Generally, a dailyregimen may be in the range of 0.2–150 μg/kg of fusion protein perkilogram of body weight. This dosage regimen is referenced to a standardlevel of biological activity which recognizes that native IL-3 generallypossesses an EC₅₀ at or about 10 picoMolar to 100 picoMolar in the AMLproliferation assay described herein. Therefore, dosages would beadjusted relative to the activity of a given fusion protein vs. theactivity of native (reference) IL-3 and it would not be unreasonable tonote that dosage regimens may include doses as low as 0.1 microgram andas high as 1 milligram per kilogram of body weight per day. In addition,there may exist specific circumstances where dosages of fusion moleculewould be adjusted higher or lower than the range of 10–200 microgramsper kilogram of body weight. These include co-administration with othercolony stimulating factor or IL-3 variant or growth factors;co-administration with chemotherapeutic drugs and/or radiation; the useof glycosylated fusion protein; and various patient-related issuesmentioned earlier in this section. As indicated above, the therapeuticmethod and compositions may also include co-administration with otherhuman factors. A non-exclusive list of other appropriate hematopoietins,CSFs, cytokines, lymphokines, hematopoietic growth factors andinterleukins for simultaneous or serial co-administration with thepolypeptides of the present invention includes GM-CSF, CSF-1, G-CSF,Meg-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF, B-cell growth factor,B-cell differentiation factor and eosinophil differentiation factor,stem cell factor (SCF) also known as steel factor or c-kit ligand, orcombinations thereof. The dosage recited above would be adjusted tocompensate for such additional components in the therapeuticcomposition. Progress of the treated patient can be monitored byperiodic assessment of the hematological profile, e.g., differentialcell count and the like.

The present invention is also directed to the following;

1. A fusion protein having the formula selected from the groupconsisting ofR₁-L-R₂, R₂-L-R₁, R₁-R₂ or R₂-R₁

-   -   wherein R₁ is a human interleukin-3 mutant polypeptide of the        Formula:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn [SEQ IDNO:1]  1               5                   10                 15 Cys XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 20                  25                 30 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa                 35                  40                 45 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 50                  55                 60 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 65                  70                 75 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 80                  85                 90 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 95                 100                105 Xaa Phe XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                110                 115                120 Xaa Xaa XaaGln Gln Thr Thr Leu Ser Leu Ala Ile Phe                125                 130wherein

-   Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;-   Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;-   Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;-   Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;-   Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn,    Thr, Ser or Val;-   Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln,    Leu, Val or Gly;-   Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Leu,    Ser, or Arg;-   Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;-   Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;-   Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;-   Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;-   Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;-   Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;-   Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or    Lys;-   Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;-   Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;-   Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;-   Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg,    Ala, Phe, Ile or Met;-   Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;-   Xaa at position 36 is Asp, Leu, or Val;-   Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;-   Xaa at position 38 is Asn, or Ala;-   Xaa at position 40 is Leu, Trp, or Arg;-   Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;-   Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val,    Glu, Phe, Tyr, Ile, Met or Ala;-   Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln,    Arg, Thr, Gly or Ser;-   Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu,    Asn, Gln, Ala or Pro;-   Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp,    Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;-   Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys,    His, Ala, Tyr, Ile, Val or Gly;-   Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;-   Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys,    Thr, Ala, Met, Val or Asn;-   Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;-   Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala,    Ile, Val, His, Phe, Met or Gln;-   Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;-   Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;-   Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or    Met;-   Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys,    His, Ala or Leu;-   Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;-   Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr,    Ala, Tyr, Phe, Val or Lys;-   Xaa at position 57 is Asn or Gly;-   Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;-   Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg;-   Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;-   Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;-   Xaa at position 62 is Asn His, Val, Arg, Pro, Thr, Asp, or Ile;-   Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;-   Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;-   Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;-   Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;-   Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or    His;-   Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;-   Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or    Leu;-   Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;-   Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp,    or Asn;-   Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;-   Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;-   Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;-   Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln,    or Leu;-   Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or    Asp;-   Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;-   Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;-   Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;-   Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;-   Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;-   Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His,    Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;-   Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;-   Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;-   Xaa at position 85 is Leu, Asn, Val, or Gln;-   Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;-   Xaa at position 87 is Leu, Ser, Trp, or Gly;-   Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;-   Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or    Ser;-   Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;-   Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;-   Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or    Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or    Arg;-   Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His,    Ala, or Pro;-   Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn,    Lys, Ser, Ala, Trp, Phe, Ile, or Tyr;-   Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;-   Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;-   Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln,    Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;-   Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser,    Phe, or His;-   Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or    Pro;-   Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu,    Asn, Ser, Ala, Gly, Ile, Leu, or Gln;-   Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;-   Xaa at position 103 is Asp, or Ser;-   Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln,    Lys, Ala, Phe, or Gly;-   Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu,    Lys, Ile, Asp, or His;-   Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;-   Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser,    Ala or Pro;-   Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;-   Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu,    Ser, Ala, or Trp;-   Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;-   Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;-   Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys,    Leu, Ile, Val or Asn;-   Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;-   Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp,    or Met;-   Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg,    Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;-   Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;-   Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;-   Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;-   Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;-   Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;-   Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile,    Tyr, or Cys;-   Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;    and which can additionally have Met- preceding the amino acid in    position 1; and wherein from 1 to 14 amino acids can be deleted from    the N-terminus and/or from 1 to 15 amino acids can be deleted from    the C-terminus; and wherein from 1 to 3 of the amino acids    designated by Xaa are different from the corresponding amino acids    of native (1–133) human interleukin-3;    -   R₂ is a colony stimulating factor selected from the following;        GM-CSF, CSF-1, G-CSF, Meg-CSF, M-CSF, erythropoietin (EPO),        IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,        IL-12, IL-13, LIF, flt3/flk2, human growth hormone, B-cell        growth factor, B-cell differentiation factor, eosinophil        differentiation factor and stem cell factor (SCF); and    -   L is a linker capable of linking R₁ to R₂.

2. The fusion protein of claim 1 wherein said human interleukin-3 mutantpolypeptide is of the Formula:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn [SEQ IDNO:2]  1               5                   10                 15 Cys XaaXaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa                 20                  25                 30 Xaa Xaa XaaXaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa                 35                  40                 45 Xaa Ile LeuMet Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa                 50                  55                 60 Phe Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu                 65                  70                 75 Xaa Xaa LeuXaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala                 80                  85                 90 Xaa Pro XaaArg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa                 95                 100                105 Xaa Phe XaaXaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu Xaa                110                 115                120 Xaa Xaa XaaGln Gln Thr Thr Leu Ser Leu Ala Ile Phe                125                 130wherein

-   Xaa at position 17 is Ser, Gly, Asp, Mete or Gln;-   Xaa at position 18 is Asn, His, or Ile;-   Xaa at position 19 is Met or Ile;-   Xaa at position 21 is Asp or Glu;-   Xaa at position 23 is Ile, Ala, Leu, or Gly;-   Xaa at position 24 is Ile, Val, or Leu;-   Xaa at position 25 is Thr, His, Gln, or Ala;-   Xaa at position 26 is His or Ala;-   Xaa at position 29 is Gln, Asn, or Val;-   Xaa at position 30 is Pro, Gly, or Gln;-   Xaa at position 31 is Pro, Asp, Gly, or Gln;-   Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;-   Xaa at position 33 is Pro or Glu;-   Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gin, Glu,    Ile, Phe, Thr or Met;-   Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val;-   Xaa at position 37 is Phe, Ser, Pro, or Trp;-   Xaa at position 38 is Asn or Ala;-   Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met,    Tyr or Arg;-   Xaa at position 44 is Asp or Glu;-   Xaa at position 45 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or    Lys;-   Xaa at position 46 is Asp, Phe, Ser, Thr, Ala, Asn Gln, Glu, His,    Ile, Lys, Tyr, Val or Cys;-   Xaa at position 50 is Glu, Ala, Asn, Ser or Asp;-   Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;-   Xaa at position 54 is Arg or Ala;-   Xaa at position 55 is Arg, Thr, Val, or Gly;-   Xaa at position 56 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu,    Thr, Val or Lys;-   Xaa at position 60 is Ala or Ser;-   Xaa at position 62 is Asn, Pro, Thr, or Ile;-   Xaa at position 63 is Arg or Lys;-   Xaa at position 64 is Ala or Asn;-   Xaa at position 65 is Val or Thr;-   Xaa at position 66 is Lys or Arg;-   Xaa at position 67 is Ser, Phe, or His;-   Xaa at position 68 is Leu, Ile, Phe, or His;-   Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;-   Xaa at position 71 is Ala, Pro, or Arg;-   Xaa at position 72 is Ser, Glu, Arg, or Asp;-   Xaa at position 73 is Ala or Leu;-   Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;-   Xaa at position 77 is Ile or Leu;-   Xaa at position 79 is Lys, Thr, Gly, Asn, Met, Arg, Ile, Gly, or    Asp;-   Xaa at position 80 is Asn, Gly, Glu, or Arg;-   Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His,    Ile, Met, Phe, Ser, Thr, Tyr or Val;-   Xaa at position 83 is Pro or Thr;-   Xaa at position 85 is Leu or Val;-   Xaa at position 87 is Leu or Ser;-   Xaa at position 88 is Ala or Trp;-   Xaa at position 91 is Ala or Pro;-   Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;-   Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser or    Thr;-   Xaa at position 96 is Pro or Tyr;-   Xaa at position 97 is Ile or Val;-   Xaa at position 98 is His, Ile, Asn, Leu, Ala, Thr, Leu, Arg, Gln,    Leu, Lys, Met, Ser, Tyr, Val or Pro;-   Xaa at position 99 is Ile, Leu, or Val;-   Xaa at position 100 is Lys, Arg, Ile, Gln, Pro, or Ser;-   Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Pro, Asn, Ile,    Leu or Tyr;-   Xaa at position 104 is Trp or Leu;-   Xaa at position 105 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys,    Ile, Asp, or His;-   Xaa at position 106 is Glu or Gly;-   Xaa at position 108 is Arg, Ala, or Ser;-   Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;-   Xaa at position 112 is Thr, Val, or Gln;-   Xaa at position 114 is Tyr or Trp;-   Xaa at position 115 is Leu or Ala;-   Xaa at position 116 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe,    Tyr or Ile;-   Xaa at position 117 is Thr or Ser;-   Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;-   Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;-   Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile,    Tyr, or Cys;-   Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;    and which can additionally have Met- preceding the amino acid in    position 1; and wherein from 1 to 14 amino acids can be deleted from    the N-terminus and/or from 1 to 15 amino acids can be deleted from    the C-terminus; and wherein from 1 to 3 of the amino acids    designated by Xaa are different from the corresponding amino acids    of native (1–133) human interleukin-3.

3. The fusion protein of claim 2 wherein said human interleukin-3 mutantpolypeptide is of the Formula:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn [SEQ IDNO:3]  1               5                   10                 15 Cys XaaXaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa                 20                  25                 30 Pro Xaa ProXaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa                 35                  40                 45 Xaa Ile LeuMet Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala                 50                  55                 60 Phe Xaa ArgXaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu                 65                 70                  75 Xaa Xaa LeuXaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala                 80                  85                 90 Xaa Pro XaaArg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa                 95                 100                105 Glu Phe XaaXaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa                 110                115                120 Xaa Xaa XaaGln Gln Thr Thr Leu Ser Leu Ala Ile Phe                125                 130wherein

-   Xaa at position 17 is Ser, Gly, Asp, or Gln;-   Xaa at position 18 is Asn, His, or Ile;-   Xaa at position 23 is Ile, Ala, Leu, or Gly;-   Xaa at position 25 is Thr, His, or Gln;-   Xaa at position 26 is His or Ala;-   Xaa at position 29 is Gln or Asn;-   Xaa at position 30 is Pro or Gly;-   Xaa at position 32 is Leu, Arg, Asn, or Ala;-   Xaa at position 34 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe,    Thr, or Met;-   Xaa at position 35 is Leu, Ala, Asn, or Pro;-   Xaa at position 38 is Asn or Ala;-   Xaa at position 42 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or    Arg;-   Xaa at position 45 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys;-   Xaa at position 46 is Asp, Phe, Ser, Gln, Glu, His, Val or Thr;-   Xaa at position 50 is Glu Asn, Ser or Asp;-   Xaa at position 51 is Asn, Arg, Pro, Thr, or His;-   Xaa at position 55 is Arg, Leu, or Gly;-   Xaa at position 56 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;-   Xaa at position 62 is Asn, Pro, or Thr;-   Xaa at position 64 is Ala or Asn;-   Xaa at position 65 is Val or Thr;-   Xaa at position 67 is Ser or Phe;-   Xaa at position 68 is Leu or Phe;-   Xaa at position 69 is Gln, Ala, Glu, or Arg;-   Xaa at position 76 is Ser, Val, Asn, Pro, or Gly;-   Xaa at position 77 is Ile or Leu;-   Xaa at position 79 is Lys, Gly, Asn, Met, Arg, Ile, or Gly;-   Xaa at position 80 is Asn, Gly, Glu, or Arg;-   Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met,    Phe, Ser, Thr, Tyr or Val;-   Xaa at position 87 is Leu or Ser;-   Xaa at position 88 is Ala or Trp;-   Xaa at position 91 is Ala or Pro;-   Xaa at position 93 is Thr, Asp, or Ala;-   Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;-   Xaa at position 98 is His, Ile, Asn, Ala, Thr, Gln, Glu, Lys, Met,    Ser, Tyr, Val or Leu;-   Xaa at position 99 is Ile or Leu;-   Xaa at position 100 is Lys or Arg;-   Xaa at position 101 is Asp, Pro, Met, Lys, Thr, His, Pro, Asn, Ile,    Leu or Tyr;-   Xaa at position 105 is Asn, Pro, Ser, Ile or Asp;-   Xaa at position 108 is Arg, Ala, or Ser;-   Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;-   Xaa at position 112 is Thr or Gln;-   Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, Tyr or Ile;-   Xaa at position 117 is Thr or Ser;-   Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;-   Xaa at position 121 is Ala, Ser, Ile, Pro, or Asp;-   Xaa at position 122 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;-   Xaa at position 123 is Ala, Met, Glu, Ser, or Leu;    and which can additionally have Met- preceding the amino acid in    position 1; and wherein from 1 to 14 amino acids can be deleted from    the N-terminus and/or from 1 to 15 amino acids can be deleted from    the C-terminus; and wherein from 1 to 3 of the amino acids    designated by Xaa are different from the corresponding amino acids    of native (1–133) human interleukin-3.

4—The fusion protein of claim 3 wherein said human interleukin-3 mutantpolypeptide is of the Formula:

-   Xaa at position 42 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;-   Xaa at position 45 is Gln, Val, Met or Asn;-   Xaa at position 46 is Asp, Ser, Gln, His or Val;-   Xaa at position 50 is Glu or Asp;-   Xaa at position 51 is Asn, Pro or Thr;-   Xaa at position 62 is Asn or Pro;-   Xaa at position 76 is Ser, or Pro;-   Xaa at position 82 is Leu, Trp, Asp, Asn Glu, His, Phe, Ser or Tyr;-   Xaa at position 95 is His, Arg, Thr, Asn or Ser;-   Xaa at position 98 is His, Ile, Leu, Ala, Gln, Lys, Met, Ser, Tyr or    Val;-   Xaa at position 100 is Lys or Arg;-   Xaa at position 101 is Asp, Pro, His, Asn, Ile or Leu;-   Xaa at position 105 is Asn, or Pro;-   Xaa at position 108 is Arg, Ala, or Ser;-   Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, or Tyr;-   Xaa at position 121 is Ala, or Ile;-   Xaa at position 122 is Gln, or Ile; and-   Xaa at position 123 is Ala, Met or Glu.

5. A fusion protein having the formula selected from the groupconsisting ofR₁-L-R₂, R₂-L-R₁, R₁-R₂ or R₂-R₁

wherein R₁ is a human interleukin-3 mutant polypeptide of the Formula:

Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa [SEQ IDNO:4]  1               5                   10                 15 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa                 20                  25                 30 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 35                  40                 45 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 50                  55                 60 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 65                  70                 75 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 80                  85                 90 Xaa Xaa PheXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 95                 100                105 Xaa Xaa XaaXaa Gln Gln                 110wherein

-   Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;-   Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln;-   Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;-   Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;-   Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn,    Thr, Ser or Val;-   Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln,    Leu, Val, or Gly;-   Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Leu,    Ser, or Arg;-   Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;-   Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala;-   Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp;-   Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala;-   Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;-   Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val;-   Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or    Lys;-   Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;-   Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;-   Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu;-   Xaa at position 20 is Leu, Val, Ser, Lys, Glu, Gln, Thr, Arg, Ala,    Phe, Ile or Met;-   Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;-   Xaa at position 22 is Asp, Leu, or Val;-   Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile;-   Xaa at position 24 is Asn, or Ala;-   Xaa at position 26 is Leu, Trp, or Arg;-   Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro;-   Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu,    Val, Glu, Phe, Tyr, Ile or Met;-   Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln,    Arg, Thr, Gly or Ser;-   Xaa at position 30 is Asp, Ser, Arg, Lys, Thr, Met, Trp, Glu, Asn,    Gln, Ala or Pro;-   Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp,    Asn, Arg, Ser, Ala, Ile, Glu, His or Trp;-   Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys,    His, Ala, Tyr, Ile, Val or Gly;-   Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His;-   Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys,    Thr, Ala, Met, Val or Asn;-   Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;-   Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala,    Ile, Val, His, Phe, Met or Gln;-   Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;-   Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr;-   Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, Met,    or;-   Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys,    His, Ala or Leu;-   Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly;-   Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr,    Ala, Tyr, Phe, Leu, Val or Lys;-   Xaa at position 43 is Asn or Gly;-   Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;-   Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg;-   Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr;-   Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;-   Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;-   Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;-   Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys;-   Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser;-   Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;-   Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or    His;-   Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;-   Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or    Leu;-   Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala;-   Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp,    or Asn;-   Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;-   Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;-   Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala;-   Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln,    or Leu;-   Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or    Asp;-   Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu;-   Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;-   Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp;-   Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;-   Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;-   Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His,    Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;-   Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met;-   Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val;-   Xaa at position 71 is Leu, Asn, Val, or Gln;-   Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys;-   Xaa at position 73 is Leu, Ser, Trp, or Gly;-   Xaa at position 74 is Ala, Lys, Arg, Val, or Trp;-   Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or    Ser;-   Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;-   Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;-   Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or    Leu;-   Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;-   Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His,    Ala or Pro;-   Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn,    Lys, Ser, Ala, Trp, Phe, Ile or Tyr;-   Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr;-   Xaa at position 83 is Ile, Val, Lys, Ala, or Asn;-   Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln,    Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;-   Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser,    Phe, or His;-   Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, Pro;-   Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu,    Asn, Ser, Ala, Gly, Ile, Leu or Gln;-   Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;-   Xaa at position 89 is Asp, or Ser;-   Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln,    Lys, Ala, Phe, or Gly;-   Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu,    Lys, Ile, Asp, or His;-   Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;-   Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser,    Ala, or Pro;-   Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;-   Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser,    Ala or Trp;-   Xaa at position 97 is Leu, Ile, Arg, Asp, or Met;-   Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;-   Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys,    Leu, Ile, Val or Asn;-   Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;-   Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp,    or Met;-   Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg,    Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;-   Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;-   Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;-   Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;-   Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln;-   Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;-   Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile,    Tyr, or Cys;-   Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;    and which can additionally have Met- or Met-Ala-preceding the amino    acid in position 1; and wherein from 1 to 3 of the amino acids    designated by Xaa are different from the corresponding native amino    acids of (1–133) human interleukin-3;    -   R₂ is a colony stimulating factor selected from the following        GM-CSF, CSF-1, G-CSF, Meg-CSF, M-CSF, erythropoietin (EPO),        IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,        IL-12, IL-13, LIF, flt3/flk2, human growth hormone, B-cell        growth factor, B-cell differentiation factor, eosinophil        differentiation factor and stem cell factor (SCF); and    -   L is a linker capable of Linking R₁ to R₂.

6. The fusion protein of claim 5 wherein said human interleukin-3 mutantpolypeptide is of the Formula:

 Asn Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa [SEQ IDNO:5]  1                5                  10                  15 XaaXaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa                 20                  25                 30 Xaa Xaa IleLeu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu                 35                  40                 45 Xaa Phe XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile                 50                  55                 60 Glu Xaa XaaLeu Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr                 65                  70                 75 Ala Xaa ProXaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa                 80                  85                 90 Xaa Xaa PheXaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu                 95                 100                105 Xaa Xaa XaaXaa Gln Gln                 110wherein

-   Xaa at position 3 is Ser, Gly, Asp, Met, or Gln;-   Xaa at position 4 is Asn, His, or Ile;-   Xaa at position 5 is Met or Ile;-   Xaa at position 7 is Asp or Glu;-   Xaa at position 9 is Ile, Ala, Leu, or Gly;-   Xaa at position 10 is Ile, Val, or Leu;-   Xaa at position 11 is Thr, His, Gln, or Ala;-   Xaa at position 12 is His or Ala;-   Xaa at position 15 is Gln, Asn, or Val;-   Xaa at position 16 is Pro, Gly, or Gln;-   Xaa at position 17 is Pro, Asp, Gly, or Gln;-   Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;-   Xaa at position 19 is Pro or Glu;-   Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu,    Ile, Phe, Thr or Met;-   Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val;-   Xaa at position 23 is Phe, Ser, Pro, or Trp;-   Xaa at position 24 is Asn or Ala;-   Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met    Tyr or Arg;-   Xaa at position 30 is Asp or Glu;-   Xaa at position 31 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or    Lys;-   Xaa at position 32 is Asp, Phe, Ser, Thr, Ala, Asn, Gln, Glu, His,    Ile, Lys, Tyr, Val or Cys;-   Xaa at position 36 is Glu, Ala, Asn, Ser or Asp;-   Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;-   Xaa at position 40 is Arg or Ala;-   Xaa at position 41 is Arg, Thr, Val, Leu, or Gly;-   Xaa at position 42 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu,    Thr, Val or Lys;-   Xaa at position 46 is Ala or Ser;-   Xaa at position 48 is Asn, Pro, Thr, or Ile;-   Xaa at position 49 is Arg or Lys;-   Xaa at position 50 is Ala or Asn;-   Xaa at position 51 is Val or Thr;-   Xaa at position 52 is Lys or Arg;-   Xaa at position 53 is Ser, Phe, or His;-   Xaa at position 54 is Leu, Ile, Phe, or His;-   Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;-   Xaa at position 57 is Ala, Pro, or Arg;-   Xaa at position 58 is Ser, Glu, Arg, or Asp;-   Xaa at position 59 is Ala or Leu;-   Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;-   Xaa at position 63 is Ile or Leu;-   Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, Gly, or    Asp;-   Xaa at position 66 is Asn, Gly, Glu, or Arg;-   Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His,    Ile, Met, Phe, Ser, Thr, Tyr or Val;-   Xaa at position 69 is Pro or Thr;-   Xaa at position 71 is Leu or Val;-   Xaa at position 73 is Leu or Ser;-   Xaa at position 74 is Ala or Trp;-   Xaa at position 77 is Ala or Pro;-   Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;-   Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser or    Thr;-   Xaa at position 82 is Pro or Tyr;-   Xaa at position 83 is Ile or Val;-   Xaa at position 84 is His, Ile, Asn, Leu, Ala, Thr, Leu, Arg, Gln,    Leu, Lys, Met, Ser, Tyr, Val or Pro;-   Xaa at position 85 is Ile, Leu, or Val;-   Xaa at position 86 is Lys, Arg, Ile, Gln, Pro, or Ser;-   Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Asn, Ile, Leu or    Tyr;-   Xaa at position 90 is Trp or Leu;-   Xaa at position 91 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys,    Ile, Asp, or His;-   Xaa at position 92 is Glu, or Gly;-   Xaa at position 94 is Arg, Ala, or Ser;-   Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;-   Xaa at position 98 is Thr, Val, or Gln;-   Xaa at position 100 is Tyr or Trp;-   Xaa at position 101 is Leu or Ala;-   Xaa at position 102 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe,    Tyr or Ile;-   Xaa at position 103 is Thr or Ser;-   Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;-   Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;-   Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile,    Tyr, or Cys;-   Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;    which can additionally have Met- or Met-Ala-preceding the amino acid    in position 1; and wherein from 1 to 3 of the amino acids designated    by Xaa are different from the corresponding amino acids of native    human interleukin-3.

7—The fusion protein of claim 6 wherein said human interleukin-3 mutantpolypeptide is of the Formula:

Asn Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa [SEQ IDNO:6]  1               5                   10                 15 Xaa ProXaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp                 20                  25                 30 Xaa Xaa IleLeu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu                 35                  40                 45 Ala Phe XaaArg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile                 50                  55                 60 Glu Xaa XaaLeu Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr                 65                  70                 75 Ala Xaa ProXaa Arg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp                 80                  85                 90 Xaa Glu PheXaa Xaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu                 95                 100                105 Xaa Xaa XaaXaa Gln Gln             110wherein

-   Xaa at position 3 is Ser, Gly, Asp, or Gln;-   Xaa at position 4 is Asn, His, or Ile;-   Xaa at position 9 is Ile, Ala, Leu, or Gly;-   Xaa at position 11 is Thr, His, or Gln;-   Xaa at position 12 is His or Ala;-   Xaa at position 15 is Gln or Asn;-   Xaa at position 16 is Pro or Gly;-   Xaa at position 18 is Leu, Arg, Asn, or Ala;-   Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe,    Thr or Met;-   Xaa at position 21 is Leu, Ala, Asn, or Pro;-   Xaa at position 24 is Asn or Ala;-   Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or    Arg;-   Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;-   Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;-   Xaa at position 36 is Glu, Asn, Ser or Asp;-   Xaa at position 37 is Asn, Arg, Pro, Thr, or His;-   Xaa at position 41 is Arg, Leu, or Gly;-   Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;-   Xaa at position 48 is Asn, Pro, or Thr;-   Xaa at position 50 is Ala or Asn;-   Xaa at position 51 is Val or Thr;-   Xaa at position 53 is Ser or Phe;-   Xaa at position 54 is Leu or Phe;-   Xaa at position 55 is Gln, Ala, Glu, or Arg;-   Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;-   Xaa at position 63 is Ile or Leu;-   Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;-   Xaa at position 66 is Asn, Gly, Glu, or Arg;-   Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met,    Phe, Ser, Thr, Tyr or Val;-   Xaa at position 73 is Leu or Ser;-   Xaa at position 74 is Ala or Trp;-   Xaa at position 77 is Ala or Pro;-   Xaa at position 79 is Thr, Asp, or Ala;-   Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;-   Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys,    Met, Ser, Tyr, Val or Leu;-   Xaa at position 85 is Ile or Leu;-   Xaa at position 86 is Lys or Arg;-   Xaa at position 87 is Asp, Pro, Met, Lys, His, Pro, Asn, Ile, Leu or    Tyr;-   Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;-   Xaa at position 94 is Arg, Ala, or Ser;-   Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;-   Xaa at position 98 is Thr or Gln;-   Xaa at position 102 is Lys, Val, Trp, or Ile;-   Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;-   Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;-   Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;-   Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;-   Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;    and which can additionally have Met- or Met-Ala-preceding the amino    acid in position 1; and wherein from 1 to 3 of the amino acids    designated by Xaa are different from the corresponding amino acids    of native (1–133) human interleukin-3.

8—The fusion protein of claim 7 wherein said human interleukin-3 mutantpolypeptide is of the Formula:

-   Xaa at position 17 is Ser, Lys, Asp, Met, Gln, or Arg;-   Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gin;-   Xaa at position 19 is Met, Arg, Gly, Ala, or Cys;-   Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;-   Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, or Val;-   Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, or Gly;-   Xaa at position 23 is Ile, Ala, Gly, Trp, Lys, Leu, Ser, or Arg;-   Xaa at position 24 is Ile, Gly, Arg, or Ser;-   Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;-   Xaa at position 26 is His, Thr, Phe, Gly, Ala, or Trp;-   Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;-   Xaa at position 28 is Lys, Leu, Gln, Gly, Pro, Val or Trp;-   Xaa at position 29 is Gln, Asn, Pro, Arg, or Val;-   Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or    Lys;-   Xaa at position 31 is Pro, Asp, Gly, Arg, Leu, or Gln;-   Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;-   Xaa at position 33 is Pro, Leu, Gln, Thr, or Glu;-   Xaa at position 34 is Leu, Gly, Ser, or Lys;-   Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, or Gln;-   Xaa at position 36 is Asp, Leu, or Val;-   Xaa at position 37 is Phe, Ser, or Pro;-   Xaa at position 38 is Asn, or Ala;-   Xaa at position 40 is Leu, Trp, or Arg;-   Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, Pro;-   Xaa at position 42 is Gly, Asp, Ser, Cys, or Ala;-   Xaa at position 42 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, or    Ser;-   Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, or    Pro;-   Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, or    Trp;-   Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, or Gly;-   Xaa at position 47 is Ile, Gly, Ser, Arg, Pro, or His;-   Xaa at position 48 is Leu, Ser, Cys, Arg, His, Phe, or Asn;-   Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;-   Xaa at position 50 is Glu, Leu, Thr, Asp, or Tyr;-   Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;-   Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;-   Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, or, Ser;-   Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, or Leu;-   Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;-   Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, or Lys;-   Xaa at position 57 is Asn or Gly;-   Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;-   Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg;-   Xaa at position 60 is Ala, Ser, Tyr, Asn, or Thr;-   Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;-   Xaa at position 62 is Asn His, Val, Arg, Pro, Thr, or Ile;-   Xaa at position 63 is Arg, Tyr, Trp, Ser, Pro, or Val;-   Xaa at position 64 is Ala, Asn, Ser, or Lys;-   Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;-   Xaa at position 66 is Lys, Ile, Val, Asn, Glu, or Ser;-   Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or    His;-   Xaa at position 68 is Leu, Val, Trp, Ser, Thr, or His;-   Xaa at position 69 is Gln, Ala, Pro, Thr, Arg, Trp, Gly, or Leu;-   Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;-   Xaa at position 71 is Ala, Met, Leu, Arg, Glu, Thr, Gln, Trp, or    Asn;-   Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;-   Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;-   Xaa at position 74 is Ile, Thr, Pro, Arg, Gly, Ala;-   Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, or    Leu;-   Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or    Asp;-   Xaa at position 77 is Ile, Ser, Arg, or Thr;-   Xaa at position 78 is Leu, Ala, Ser, Glu, Gly, or Arg;-   Xaa at position 79 is Lys, Thr, Gly, Asn, Met, Ile, or Asp;-   Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, or Arg;-   Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, or Lys;-   Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, or Asp;-   Xaa at position 83 is Pro, Thr, Trp, Arg, or Met;-   Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;-   Xaa at position 85 is Leu, Asn, or Gln;-   Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;-   Xaa at position 87 is Leu, Ser, Trp, or Gly;-   Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;-   Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, or Asn;-   Xaa at position 90 is Ala, Ser, Asp, Ile, or Met;-   Xaa at position 91 is Ala, Ser, Thr, Phe, Leu, Asp, or His;-   Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, or Leu;-   Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;-   Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, or Pro;-   Xaa at position 95 is His, Gln, Pro, Val, Leu, Thr or Tyr;-   Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;-   Xaa at position 97 is Ile, Lys, Ala, or Asn;-   Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, or Pro;-   Xaa at position 99 is Ile, Arg, Asp, Pro, Gln, Gly, Phe, or His;-   Xaa at position 100 is Lys, Tyr, Leu, His, Ile, Ser, Gln, or Pro;-   Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, or    Gln;-   Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;-   Xaa at position 103 is Asp, or Ser;-   Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln,    Lys, Ala, Phe, or Gly;-   Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu,    Lys, Ile, or His;-   Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;-   Xaa at position 108 is Arg, Asp, Leu, Thr, Ile, or Pro;-   Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly.    Materials and Methods for Fusion Molecule Expression in E. coli

Unless noted otherwise, all specialty chemicals are obtained from SigmaCo., (St. Louis, Mo.). Restriction endonucleases, T4 poly-nucleotideskinase, E. coli DNA polymerase I large fragment (Klenow) and T4 DNAligase are obtained from New England Biolabs (Beverly, Mass.).

Escherichia coli Strains

Strain JM101: delta (pro lac), supE, thi, F′ (traD36, rpoAB, lacI-Q,lacZdeltaM15) (Messing, 1979). This Strain can be obtained from theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, accession number 33876. MON 105 (W3110 rpoH358) isa derivative of W3110 (Bachmann, 1972) and has been assigned ATCCaccession number 55204. Strain GM48: dam-3, dcm-6, gal, ara, lac, thr,leu, topA, tsx (Marinus, 1973) is used to make plasmid DNA that is notmethylated at the sequence GATC.

Genes and Plasmids

The gene used for hIL-3 production in E. coli is obtained from BritishBiotechnology Incorporated, Cambridge, England, catalogue number BBG14.This gene is carried on a pUC based plasmid designated pP0518. Manyother human CSF genes can be obtained from R&D Systems, Inc. (Mann,Minn.) including IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7,IL-8, G-CSF, GM-CSF and LIF.

The plasmids used for production hIL-3 in E. coli contain geneticelements whose use has been described (Olins et al., 1988; Olins andRangwala, 1990). The replicon used is that of pBR327 (Covarrubias, etal., 1981) which is maintained at a copy number of about 100 in the cell(Soberon et al., 1980). A gene encoding the beta-lactamase protein ispresent on the plasmids. This protein confers ampicillin resistance onthe cell. This resistance serves as a selectable phenotype for thepresence of the plasmid in the cell.

For cytoplasmic expression vectors the transcription promoter is derivedfrom the recA gene of E. coli (Sancar et al., 1980). This promoter,designated precA, includes the RNA polymerase binding site and the lexArepressor binding site (the operator). This segment of DNA provides highlevel transcription that is regulated even when the recA promoter is ona plasmid with the pBR327 origin of replication (Olins et al., 1988)incorporated herein by reference.

The ribosome binding site used is that from gene 10 of phage T7 (Olinset al., 1988). This is encoded in a 100 base pair (bp) fragment placedadjacent to precA. In the plasmids used herein, the recognition sequencefor the enzyme NcoI (CCATGG) follows the g10-L. It is at this NcoI sitethat the hIL-3 genes are joined to the plasmid. It is expected that thenucleotide sequence at this junction will be recognized in mRNA as afunctional start site for translation (Olins et al., 1988). The hIL-3genes used were engineered to have a HindIII recognition site (AAGCTT)downstream from the coding sequence of the gene. At this HindIII site isa 514 base pair RsaI fragment containing the origin of replication ofthe single stranded phage f1 (Dente et al., 1983; Olins, et al., 1990)both incorporated herein by reference. A plasmid containing theseelements is pMON2341. Another plasmid containing these elements ispMON5847 which has been deposited at the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 under theaccession number ATCC 68912.

In secretion expression plasmids the transcription promoter is derivedfrom the ara B, A, and D genes of E. coli (Greenfield et al., 1978).This promoter is designated pAraBAD and is contained on a 323 base pairSacII, BglII restriction fragment. The LamB secretion leader (Wong etal., 1988, Clement et al., 1981) is fused to the N-terminus of the hIL-3gene at the recognition sequence for the enzyme NcoI (5′CCATGG3′). ThehIL-3 genes used were engineered to have a HindIII recognition site(5′AAGCTT3′) following the coding sequence of the gene.

Recombinant DNA Methods

Synthetic Gene Assembly

The hIL-3 variant genes and other CSF genes can be constructed by theassembly of synthetic oligonucleotides. Synthetic oligonucleotides aredesigned so that they would anneal in complementary pairs, withprotruding single stranded ends, and when the pairs are properlyassembled would result in a DNA sequence that encoded a portion of thedesired gene. Amino acid substitutions in the hIL-3 gene are made bydesigning the oligonucleotides to encode the desired substitutions. Thecomplementary oligonucleotides are annealed at concentration of 1picomole per microliter in ligation buffer plus 50 mM NaCl. The samplesare heated in a 100 ml beaker of boiling water and permitted to coolslowly to room temperature. One picomole of each of the annealed pairsof oligonucleotides are ligated with approximately 0.2 picomoles ofplasmid DNA, digested with the appropriate restriction enzymes, inligation buffer (25 mM Tris pH 8.0, 10 mM MgCl₂, 10 mM dithiothreitol, 1mM ATP, 2 mM spermidine) with T4 DNA ligase obtained from New EnglandBiolabs (Beverly, Mass.) in a total volume of 20 μl at room temperatureovernight.

Polymerase Chain Reaction

Polymerase Chain Reaction (hereafter referred to as PCR) techniques(Saiki, 1985) used the reagent kit and thermal cycler from Perkin-ElmerCetus (Norwalk, Conn.). PCR is based on a thermostable DNA polymerasefrom Thermus aquaticus. The PCR technique is a DNA amplification methodthat mimics the natural DNA replication process in that the number ofDNA molecules doubles after each cycle, in a way similar to in vivoreplication. The DNA polymerase mediated extension is in a 5′ to 3′direction. The term “primer” as used herein refers to an oligonucleotidesequence that provides an end to which the DNA polymerase can addnucleotides that are complementary to a nucleotide sequence. The latternucleotide sequence is referred to as the “template”, to which theprimers are annealed. The amplified PCR product is defined as the regioncomprised between the 5′ ends of the extension primers. Since theprimers have defined sequences, the product will have discrete ends,corresponding to the primer sequences. The primer extension reaction iscarried out using 20 picomoles (pmoles) of each of the oligonucleotidesand 1 picogram of template plasmid DNA for 35 cycles (1 cycle is definedas 94 degrees C. for one minute, 50 degrees C. for two minutes and 72degrees for three minutes.). The reaction mixture is extracted with anequal volume of phenol/chloroform (50% phenol and 50% chloroform, volumeto volume) to remove proteins. The aqueous phase, containing theamplified DNA, and solvent phase are separated by centrifugation for 5minutes in a microcentrifuge (Model 5414 Eppendorf Inc, Fremont Calif.).To precipitate the amplified DNA the aqueous phase is removed andtransferred to a fresh tube to which is added 1/10 volume of 3M NaOAc(pH 5.2) and 2.5 volumes of ethanol (100% stored at minus 20 degreesC.). The solution is mixed and placed on dry ice for 20 minutes. The DNAis pelleted by centrifugation for 10 minutes in a microcentrifuge andthe solution is removed from the pellet. The DNA pellet is washed with70% ethanol, ethanol removed and dried in a speedvac concentrator(Savant, Farmingdale, N.Y.). The pellet is resuspended in 25 microlitersof TE (20 mM Tris-HCl pH 7.9, 1 mM EDTA). Alternatively the DNA isprecipitated by adding equal volume of 4M NH₄OAc and one volume ofisopropanol [Treco et al., (1988)]. The solution is mixed and incubatedat room temperature for 10 minutes and centrifuged. These conditionsselectively precipitate DNA fragments larger than ˜20 bases and are usedto remove oligonucleotide primers. One quarter of the reaction isdigested with restriction enzymes [Higuchi, (1989)] an on completionheated to 70 degrees C. to inactivate the enzymes.

Recovery of Recombinant Plasmids from Ligation Mixes

E. coli JM101 cells are made competent to take up DNA. Typically, 20 to100 ml of cells are grown in LB medium to a density of approximately 150Klett units and then collected by centrifugation. The cells areresuspended in one half culture volume of 50 mM CaCl₂ and held at 4° C.for one hour. The cells are again collected by centrifugation andresuspended in one tenth culture volume of 50 mM CaCl₂. DNA is added toa 150 microliter volume of these cells, and the samples are held at 4°C. for 30 minutes. The samples are shifted to 42° C. for one minute, onemilliliter of LB is added, and the samples are shaken at 37° C. for onehour. Cells from these samples are spread on plates containingampicillin to select for transformants. The plates are incubatedovernight at 37° C. Single colonies are picked, grown in LB supplementedwith ampicillin overnight at 37° C. with shaking. From these culturesDNA is isolated for restriction analysis.

Culture Medium

LB medium (Maniatis et al., 1982) is used for growth of cells for DNAisolation. M9 minimal medium supplemented with 1.0% casamino acids, acidhydrolyzed casein, Difco (Detroit, Mich.) is used for cultures in whichrecombinant fusion molecule is produced. The ingredients in the M9medium are as follows: 3 g/liter KH₂PO₄, 6 g/l Na₂HPO₄, 0.5 g/l NaCl, 1g/l NH₄Cl, 1.2 mM MgSO₄, 0.025 mM CaCl₂, 0.2% glucose (0.2% glycerolwith the AraBAD promoter), 1% casamino acids, 0.1 ml/l trace minerals(per liter 108 g FeCl₃.6H₂O, 4.0 g ZnSO₄.7H₂O, 7.0 CoCl₂.2H₂O, 7.0 gNa₂MoO₄.2H₂O, 8.0 g CuSO₄.5H₂O, 2.0 g H₃BO₃, 5.0 g MnSO₄.2O, 100 mlconcentrated HCl). Bacto agar is used for solid media and ampicillin isadded to both liquid and solid LB media at 200 micrograms permilliliter.

Production of Fusion Molecules in E. coli with Vectors Employing therecA Promoter

E. coli strains harboring the plasmids of interest are grown at 37° C.in M9 plus casamino acids medium with shaking in a Gyrotory water bathModel G76 from New Brunswick Scientific (Edison, N.J.). Growth ismonitored with a Klett Summerson meter (green 54 filter), Klett Mfg. Co.(New York, N.Y.). At a Klett value of approximately 150, an aliquot ofthe culture (usually one milliliter) is removed for protein analysis. Tothe remaining culture, nalidixic acid (10 mg/ml) in 0.1 N NaOH is addedto a final concentration of 50 μg/ml. The cultures are shaken at 37° C.for three to four hours after addition of nalidixic acid. A high degreeof aeration is maintained throughout the bacterial growth in order toachieve maximal production of the desired gene product. The cells areexamined under a light microscope for the presence of refractile bodies(RBs). One milliliter aliquots of the culture are removed for analysisof protein content.

Fractionation of E. coli Cells Producing Fusion Proteins in theCytoplasm

The first step in purification of the fusion molecules is to sonicatethe cells. Aliquots of the culture are resuspended from cell pellets insonication buffer: 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.1 mMPMSF. These resuspended cells are subjected to several repeatedsonication bursts using the microtip from a Sonicator cell disrupter,Model W-375 obtained from Heat Systems-Ultrasonics Inc. (Farmingdale,N.Y.). The extent of sonication is monitored by examining thehomogenates under a light microscope. When nearly all of the cells arebroken, the homogenates are fractionated by centrifugation. The pellets,which contain most of the refractile bodies, are highly enriched forfusion proteins.

Methods: Extraction, Refolding and Purification of Fusion MoleculesExpressed as Refractile Bodies in E. coli.

These fusion proteins can be purified by a variety of standard methods.Some of these methods are described in detail in Methods in Enzymology,Volume 182 ‘Guide to Protein Purification’, edited by Murray Deutscher,Academic Press, San Diego, Calif. (1990).Fusion proteins which are produced as insoluble inclusion bodies in E.coli can be solubilized in high concentrations of denaturant, such asGuanidine HCl or Urea including dithiothreitol or beta mercaptoethanolas a reducing agent. Folding of the protein to an active conformationmay be accomplished via sequential dialysis to lower concentrations ofdenaturant without reducing agent.In some cases the folded proteins can be affinity purified usingaffinity reagents such as mAbs or receptor subunits attached to asuitable matrix. Alternatively, (or in addition) purification can beaccomplished using any of a variety of chromatographic methods such as:ion exchange, gel filtration or hydrophobic chromatography or reversedphase HPLC.hIL-3 Sandwich Elisa

The fusion protein concentrations can be determined using a sandwichELISA based on an appropriate affinity purified antibody. Microtiterplates (Dynatech Immulon II) are coated with 150 μl goat-anti-rhIL-3 ata concentration of approximately 1 μg/ml in 100 mM NaHCO3, pH 8.2.Plates are incubated overnight at room temperature in a chambermaintaining 100% humidity. Wells are emptied and the remaining reactivesites on the plate are blocked with 200 μl of solution containing 10 mMPBS, 3% BSA and 0.05% Tween 20, pH 7.4 for 1 hour at 37° C. and 100%humidity. Wells are emptied and washed 4× with 150 mM NaCl containing0.05% Tween 20 (wash buffer). Each well then receives 150 μl of dilutionbuffer (10 mM PBS containing 0.1% BSA, 0.01% Tween 20, pH 7.4),containing rhIL-3 standard, control, sample or dilution buffer alone. Astandard curve is prepared with concentrations ranging from 0.125 ng/mlto 5 ng/ml using a stock solution of rhIL-3 (concentration determined byamino acid composition analysis). Plates are incubated 2.5 hours at 37°C. and 100% humidity. Wells are emptied and each plate is washed 4× withwash buffer. Each well then received 150 μl of an optimal dilution (asdetermined in a checkerboard assay format) of goat anti-rhIL-3conjugated to horseradish peroxidase. Plates are incubated 1.5 hours at37° C. and 100% humidity. Wells are emptied and each plate is washed 4×with wash buffer. Each well then received 150 ul of ABTS substratesolution (Kirkegaard and Perry). Plates are incubated at roomtemperature until the color of the standard wells containing 5 ng/mlrhIL-3 had developed enough to yield an absorbance between 0.5–1.0 whenread at a test wavelength of 410 nm and a reference wavelength of 570 nmon a Dynatech microtiter plate reader. Concentrations of immunoreactiverhIL-3 in unknown samples are calculated from the standard curve usingsoftware supplied with the plate reader.

AML Proliferation Assay for Bioactive Human Interleukin-3

The factor-dependent cell line AML 193 was obtained from the AmericanType Culture Collection (ATCC, Rockville, Md.). This cell line,established from a patient with acute myelogenous leukemia, is a growthfactor dependent cell line which displayed enhanced growth in GM-CSFsupplemented medium (Lange, B., et al., (1987); Valtieri, M., et al.,(1987). The ability of AML 193 cells to proliferate in the presence ofhuman IL-3 has also been documented. (Santoli, D., et al., (1987)). Acell line variant was used, AML 193 1.3, which was adapted for long termgrowth in IL-3 by washing out the growth factors and starving thecytokine dependent AML 193 cells for growth factors for 24 hours. Thecells are then replated at 1×10⁵ cells/well in a 24 well plate in mediacontaining 100 U/ml IL-3. It took approximately 2 months for the cellsto grow rapidly in IL-3. These cells are maintained as AML 193 1.3thereafter by supplementing tissue culture medium (see below) with humanIL-3.

AML 193 1.3 cells are washed 6 times in cold Hanks balanced saltsolution (HBSS, Gibco, Grand Island, N.Y.) by centrifuging cellsuspensions at 250× g for 10 minutes followed by decantation ofsupernatant. Pelleted cells are resuspended in HBSS and the procedure isrepeated until six wash cycles are completed. Cells washed six times bythis procedure are resuspended in tissue culture medium at a densityranging from 2×10⁵ to 5×10⁵ viable cells/ml. This medium is prepared bysupplementing Iscove's modified Dulbecco's Medium (IMDM, Hazleton,Lenexa, Kans.) with albumin, transferrin, lipids and 2-mercaptoethanol.Bovine albumin (Boehringer-Mannheim, Indianapolis, Ind.) is added at 500μg/ml; human transferrin (Boehringer-Mannheim, Indianapolis, Ind.) isadded at 100 μg/ml; soybean lipid (Boehringer-Mannheim, Indianapolis,Ind.) is added at 50 μg/ml; and 2-mercaptoethanol (Sigma, St. Louis,Mo.) is added at 5×10⁻⁵ M.

Serial dilutions of human interleukin-3 or fusion protein (hIL-3 mutein)are made in triplicate series in tissue culture medium supplemented asstated above in 96 well Costar 3596 tissue culture plates. Each wellcontained 50 μl of medium containing interleukin-3 or fusion proteinonce serial dilutions are completed. Control wells contained tissueculture medium alone (negative control). AML 193 1.3 cell suspensionsprepared as above are added to each well by pipetting 50 μl (2.5×10⁴cells) into each well. Tissue culture plates are incubated at 37° C.with 5% CO₂ in humidified air for 3 days. On day 3, 0.5 μCi ³H-thymidine(2 Ci/mM, New England Nuclear, Boston, Mass.) is added in 50 μl oftissue culture medium. Cultures are incubated at 37° C. with 5% CO₂ inhumidified air for 18–24 hours. Cellular DNA is harvested onto glassfilter mats (Pharmacia LKB, Gaithersburg, Md.) using a TOMTEC cellharvester (TOMTEC, Orange, Conn.) which utilized a water wash cyclefollowed by a 70% ethanol wash cycle. Filter mats are allowed to air dryand then placed into sample bags to which scintillation fluid(Scintiverse II, Fisher Scientific, St. Louis, Mo. or BetaPlateScintillation Fluid, Pharmacia LKB, Gaithersburg, Md.) is added. Betaemissions of samples from individual tissue culture wells are counted ina LKB Betaplate model 1205 scintillation counter (Pharmacia LKB,Gaithersburg, Md.) and data is expressed as counts per minute of³H-thymidine incorporated into cells from each tissue culture well.Activity of each human interleukin-3 preparation or fusion proteinpreparation is quantitated by measuring cell proliferation (³H-thymidineincorporation) induced by graded concentrations of interleukin-3 orfusion protein. Typically, concentration ranges from 0.05 pM–10⁵ pM arequantitated in these assays. Activity is determined by measuring thedose of interleukin-3 or fusion molecule which provides 50% of maximalproliferation [EC₅₀=0.5×(maximum average counts per minute of³H-thymidine incorporated per well among triplicate cultures of allconcentrations of interleukin-3 tested—background proliferation measuredby ³H-thymidine incorporation observed in triplicate cultures lackinginterleukin-3]. This EC₅₀ value is also equivalent to 1 unit ofbioactivity. Every assay is performed with native interleukin-3 as areference standard so that relative activity levels could be assigned.

Methylcellulose Assay

This assay provides a reasonable approximation of the growth activity ofcolony stimulating factors to stimulate normal bone marrow cells toproduce different types of hematopoietic colonies in vitro (Bradley etal., 1966, Pluznik et al., 1965).

Methods

Approximately 30 ml of fresh, normal, healthy bone marrow aspirate areobtained from individuals. Under sterile conditions samples are diluted1:5 with a 1×PBS (#14040.059 Life Technologies, Gaithersburg, Md.)solution in a 50 ml conical tube (#25339-50 Corning, Corningn Md.).Ficoll (Histopaque-1077 Sigma H-8889) is layered under the dilutedsample and centrifuged, 300× g for 30 min. The mononuclear cell band isremoved and washed two times in 1×PBS and once with 1% BSA PBS (CellProCo., Bothel, Wash.). Mononuclear cells are counted and CD34+ cells areselected using the Ceprate LC (CD34) Kit (CellPro Co., Bothel, Wash.)column. This fractionation is performed since all stem and progenitorcells within the bone marrow display CD34 surface antigen. Alternativelywhole bone marrow or peripheral blood may be used.Cultures are set up in triplicate wells with a final volume of 0.1 ml in48 well tissue culture plates (#3548 CoStar, Cambridge, Mass.). Culturemedium is purchased from Terry Fox Labs. (HCC-4330 medium (Terry FoxLabs, Vancouver, B.C., Canada)). 600–1000 CD34+cells are added per well.Native IL-3 and fusion molecule are added to give final concentrationsranging from 0.01 nM-10 nm. G-CSF and GM-CSF and C-Kit ligand are addedat a final concentration of 0.1 nm. Native IL-3 and fusion molecules aresupplied in house. C-Kit Ligand (#255-CS), G-CSF (#214-CS) and GM-CSF(#215-GM) are purchased from R&D Systems (Minneapolis, Minn.). Culturesare resuspended using an Eppendorf repeater and 0.1 ml is dispensed perwell. Control (baseline response) cultures received no colonystimulating factors. Positive control cultures received conditionedmedia (PHA stimulated human cells:Terry Fox Lab. H2400). Cultures areincubated at 37° C., 5% CO₂ in humidified air.Hematopoietic colonies which are defined as greater than 50 cells arecounted on the day of peak response (days 10–11) using a Nikon invertedphase microscope with a 40× objective combination. Groups of cellscontaining fewer than 50 cells are referred to as clusters.Alternatively colonies can be identified by spreading the colonies on aslide and stained or they can be picked, resuspended and spun ontocytospin slides for staining.Human Cord Blood Hemopoietic Growth Factor Assays

Bone marrow cells are traditionally used for in vitro assays ofhematopoietic colony stimulating factor (CSF) activity. However, humanbone marrow is not always available, and there is considerablevariability between donors. Umbilical cord blood is comparable to bonemarrow as a source of hematopoietic stem cells and progenitors(Broxmeyer et al., 1992; Mayani et al., 1993). In contrast to bonemarrow, cord blood is more readily available on a regular basis. Thereis also a potential to reduce assay variability by pooling cellsobtained fresh from several donors, or to create a bank of cryopreservedcells for this purpose. By modifying the culture conditions, and/oranalyzing for lineage specific markers, it should be possible to assayspecifically for granulocyte/macrophage colonies (CFU-GM), formegakaryocyte CSF activity, or for high proliferative potential colonyforming cell (HPP-CFC) activity.

Methods

Mononuclear cells (MNC) are isolated from cord blood within 24 hrs ofcollection, using a standard density gradient (1.077 g/ml Histopaque).Cord blood MNC have been further enriched for stem cells and progenitorsby several procedures, including immunomagnetic selection for CD14−,CD34+ cells; panning for SBA−, CD34+ fraction using coated flasks fromApplied Immune Science (Santa Clara, Calif.); and CD34+ selection usinga CellPro (Bothell, Wash.) avidin column. Either freshly isolated orcryopreserved CD34+ cell enriched fractions are used for the assay.Duplicate cultures for each serial dilution of sample (concentrationrange from 1 pm to 1204 pm) are prepared with 1×104 cells in 1 ml of0.9% methocellulose containing medium without additional growth factors(Methocult H4230 from Stem Cell Technologies, Vancouver, BC.). In someexperiments, Methocult H4330 containing erythropoietin (EPO) was usedinstead of Methocult H4230, or Stem Cell Factor (SCF), 50 ng/ml(Biosource International, Camarillo, Calif.) was added. After culturingfor 7–9 days, colonies containing >30 cells are counted. In order torule out subjective bias in scoring, assays are scored blind.

IL-3 Mediated Sulfidoleukotriene Release from Human Mononuclear Cells

The following assay is used to measure IL-3 mediated sulfidoleukotrienerelease from human mononuclear cells.

Heparin-containing human blood is collected and layered onto an equalvolume of Ficoll-Paque (Pharmacia # 17-0840-02) ready to use medium(density 1.077 g/ml.). The Ficoll is warmed to room temperature prior touse and clear 50 ml polystyrene tubes are utilized. The Ficoll gradientis spun at 300× g for 30 minutes at room temperature using a H1000Brotor in a Sorvall RT6000B refrigerated centrifuge. The band containingthe mononuclear cells is carefully removed, the volume adjusted to 50mls with Dulbecco's phosphate-buffered saline (Gibco Laboratories cat. #310-4040PK), spun at 400× g for 10 minutes at 4° C. and the supernatantis carefully removed. The cell pellet is washed twice with HA Buffer [20mM Hepes (Sigma # H-3375), 125 mM NaCl (Fisher # S271-500), 5 mM KCl(Sigma # P-9541), 0.5 mM glucose (Sigma # G-5000), 0.025% Human SerumAlbumin (Calbiochem # 126654) and spun at 300×g, 10 min., 4° C. Thecells are resuspended in HACM Buffer (HA buffer supplemented with 1 mMCaCl₂ (Fisher # C79-500) and 1 mM MgCl2 (Fisher # M-33) at aconcentration of 1×106 cells/ml and 180 μl are transferred into eachwell of 96 well tissue culture plates. The cells are allowed toacclimate at 37° C. for 15 minutes. The cells are primed by adding 10μls of a 20× stock of various concentrations of cytokine to each well(typically 100000, 20000, 4000, 800, 160, 32, 6.4, 1.28, 0 fM IL3). Thecells are incubated for 15 minutes at 37° C. Sulfidoleukotriene releaseis activated by the addition of 10 μls of 20×(1000 nM) fmet-leu-phe(Calbiochem # 344252) final concentration 50 nM FMLP and incubated for10 minutes at 37° C. The plates are spun at 350× g at 4° C. for 20minutes. The supernatants are removed and assayed forsulfidoleukotrienes using Cayman's Leukotriene C4 EIA kit (Cat. #420211)according to manufacturers', directions. Native hIL-3 is run as astandard control in each assay.

Further details known to those skilled in the art may be found in T.Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory (1982) and references cited therein, incorporatedherein by reference; and in J. Sambrook, et al., Molecular Cloning, ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1989) andreferences cited therein, incorporated herein by reference.

Additional details on the IL-3 variants of the present invention may befound in co-pending U.S. Patent Application Ser. No. PCT/US93/11198which is hereby incorporated by reference in its entirety as if writtenherein.

Additional details on how to make the fusion protein can be found in WO92/04455 and WO 91/02754.

Additional details about the CSFs and the variants thereof can be foundin U.S. Pat. Nos. 4,810,643, 5,218,092 and E.P. Application 02174004.

All references, patents or applications cited herein are incorporated byreference in their entirety as if written herein.

The following examples will illustrate the invention in greater detailalthough it will be understood that the invention is not limited tothese specific examples.

EXAMPLE 1

Construction of Expression Plasmid for Fusion Molecules

Construction of a plasmid encoding a fusion protein composed of the IL-3variant protein found in the plasmid, pMON13252 (U.S. Patent ApplicationSer. No. PCT/US93/11198), followed by a factor Xa proteolytic cleavagesite, followed by murine IgG 2b hinge region, in which the cysteins havereplaced with serines, as the polypeptide linker sequence between thetwo proteins of the fusion and followed by G-CSF. The plasmid,pMON13252, is digested with EcoRI (which is internal in the IL-3 variantgene) and HindIII (which is after the stop codons for the IL-3 variant)and the 3900 base pair EcoRI, HindIII restriction fragment is purified.The genetic elements derived from pMON13252 are the beta-lactamase gene(AMP), pBR327 origin of replication, recA promoter, g10L ribosomebinding site, the bases encoding amino acids 15–105 of (15–125) IL-3variant gene, and phage f1 origin of replication. Pairs of complementarysynthetic oligonucleotides are designed to replace the portion of theIL-3 variant gene after the EcoRI site (bases encoding amino acids106–125), DNA sequence encoding the factor Xa cleavage site, DNAsequence encoding the polypeptide linker and AflIII restriction site toallow for cloning of the second gene in the fusion. When properlyassembled the oligonucleotides results in a DNA sequence, encoding theabove mentioned components in-frame, with EcoRI and HindIII restrictionends. Within this DNA sequence unique restriction sites are also createdto allow for the subsequent replacement of specific regions with asequence that has similar function (eg. alternative polypeptide linkerregion). A unique SnaBI restriction site is created at the end of the13252 gene which allows for the cloning of other genes in the C-terminusposition of the fusion. A unique XmaI site is created between sequenceencoding the factor Xa cleavage site and the region encoding thepolypeptide linker. A unique AflIII site is created after the linkerregion that allows for the cloning of the N-terminal protein of thefusion. The 3900 base pair fragment from pMON13252 is ligated with theassembled oligonucleotides and transformed into an appropriate E. colistrain. The resulting clones are screened by restriction analysis andDNA sequenced to confirm that the desired DNA sequence are created. Theresulting plasmid is used as an intermediate into which other genes canbe cloned as a NcoI, HindIII fragment into the AflIII and HindIII sitesto create the desired fusion. The overhangs created by NcoI and AflIIIare compatible but the flanking sequence of the restriction recognitionsites are different. The NcoI and AflIII sites are lost as a result ofthe cloning. The above mentioned restrictions site are used as examplesand are not limited to those described. Other unique restriction sitemay also be engineered which serve the function of allowing the regionsto be replaced. The plasmid encoding the resulting fusion is DNAsequenced to confirm that the desired DNA sequence is obtained. OtherIL-3 variant genes or other colony stimulating factor genes can bealtered in a similar manner by genetic engineering techniques to createthe appropriate restriction sites which would allow for cloning eitherinto the C-terminal or N-terminal position of the fusion constructdescribed above. Likewise alternative peptidase cleavage sites orpolypeptide linkers can be engineered into the fusion plasmids.

EXAMPLE 2

Expression, Extraction, Refolding and Purification of Fusion ProteinsExpressed as Refractile Bodies in E. coli

E. coli strains harboring the plasmids of interest are grown overnightat 37° C. and diluted the following morning, approximately 1/50, infresh M9 plus casamino acids medium. The culture is grown at 37° C. forthree to four hours to mid-log (OD600=˜1) with vigorous shaking.Nalidixic acid (10 mg/ml) in 0.1 N NaOH is added to a finalconcentration of 50 μg/ml. The cultures are grown at 37° C. for three tofour hours after the addition of nalidixic acid. A high degree ofaeration is maintained throughout the bacterial growth in order toachieve maximal production of the desired fusion protein. In cases werethe fusion proteins are produced as insoluble inclusion bodies in E.coli the cells are examined under a light microscope for the presence ofretractile bodies (RBs).

The first step in purification of the fusion molecules is to sonicatethe cells. Aliquots of the culture are resuspended from cell pellets insonication buffer: 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.1 mMPMSF. These resuspended cells are subjected to several repeatedsonication bursts using the microtip from a Sonicator cell disrupter,Model W-375 obtained from Heat Systems-Ultrasonics Inc. (Farmingdale,N.Y.). The extent of sonication is monitored by examining thehomogenates under a light microscope. When nearly all of the cells arebroken, the homogenates are fractionated by centrifugation. The pellets,which contain most of the refractile bodies, are highly enriched forfusion proteins.

Fusion proteins which are produced as insoluble inclusion bodies in E.coli can be solubilized in high concentrations of denaturant, such asGuanidine HCl or Urea including dithiothreitol or beta mercaptoethanolas a reducing agent. Folding of the protein to an active conformationmay be accomplished via sequential dialysis to lower concentrations ofdenaturant without reducing agent.

In some cases the folded proteins can be affinity purified usingaffinity reagents such as mAbs or receptor subunits attached to asuitable matrix. Alternatively, (or in addition) purification can beaccomplished using any of a variety of chromatographic methods such as:ion exchange, gel filtration or hydrophobic chromatography or reversedphase HPLC. These and other protein purification methods are describedin detail in Methods in Enzymology, Volume 182 ‘Guide to ProteinPurification’ edited by Murray Deutscher, Academic Press, San Diego,Calif. (1990).

EXAMPLE 3

Determination of the In Vitro Activity of Fusion Proteins

The protein concentration of the fusion protein can be determined usinga sandwich ELISA based on an affinity purified polyclonal antibody.Alternatively the protein concentration can be determined by amino acidcomposition. The bioactivity of the fusion molecule can be determined ina number of in vitro assays compared with native IL-3, the IL-3 variantor G-CSF alone or together. One such assay is the AML-193 cellproliferation assay. AML-193 cells respond to IL-3 and G-CSF whichallows for the combined bioactivity of the IL-3 variant/G-CSF fusion tobe determined. In addition other factor dependent cell lines, such as32D which is a murine IL-3 dependent cell line, may be used. Theactivity of IL-3 is species specific whereas G-CSF is not, therefor thebioactivity of the G-CSF component of the IL-3 variant/G-CSF fusion canbe determined independently. The methylcellulose assay can be used todetermine the effect of the IL-3 variant/G-CSF fusion protein on theexpansion of the hematopoietic progenitor cells and the pattern of thedifferent types of hematopoietic colonies in vitro. The methylcelluloseassay can also provide an estimate of precursor frequency since onemeasures the frequency of progenitors per 100,000 input cells.Long-term, stromal dependent cultures have been used to delineateprimitive hematopoietic progenitors and stem cells. This assay can beused to determine whether the fusion protein stimulates the expansion ofvery primitive progenitors and/or stem cells. In addition, limitdilution cultures can be performed which will indicate the frequency ofprimitive progenitors stimulated by the fusion molecules.

The factor Xa cleavage site is useful to cleave the fusion protein afterit is purified and re-folded to separate the IL-3 and G-CSF componentsof the fusion. After cleavage with factor Xa the IL-3 and G-CSFcomponents of the fusion can be purified to homogeneity and assayedseparately to demonstrate that both components are in an activeconformation after being expressed, refolded and purified as a fusion.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included-within the scope of the appended claims.

Amino acids are shown herein by standard one letter or three letterabbreviations as follows:

Abbreviated Designation Amino Acid A Ala Alanine C Cys Cysteine D AspAspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine HHis Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M MetMethionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg ArginineS Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y TyrTyrosine

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1. A fusion protein comprising a human interleukin-3 variant polypeptidehaving increased cell proliferative activity compared to native hIL-3,wherein the variations from the native hIL-3 amino acid sequencecomprises the replacement of one or both of the residues at positions101 and 116 of said native hIL-3 sequence by other amino acids andoptionally, the deletion of from 1 to 14 amino acids from the N-terminusand/or deletion of from 1 to 15 amino acids from the C-terminus of saidhuman interleukin-3 variant polypeptide.
 2. The fusion protein of claim1, wherein said human interleukin-3 variant polypeptide has from 1 to 14amino acids are deleted from the N-terminus and/or from 1 to 15 aminoacids are deleted from the C-terminus of said human interluekin-3variant polypeptide.
 3. A fusion protein comprising a polypeptide havinga sequence selected from the group consisting of: R₁-L-R₂, R₂-L-R₁,R₁-R₂, R₂-L-R₁, Met-Ala-R₁-L-R₂, Met-Ala-R₂-L-R₁, Met-Ala-R₁-R₂,Met-Ala-R₂-R₁, Met-R₁-L-R₂, Met-R₂-L-R₁, Met-R₁-R₂, Met-R₂-R₁,Ala-R₁-L-R₂, Ala-R₂-L-R₁, Ala-R₁-R₂ and Ala-R₂-R₁; wherein R₁ is a humaninterleukin-3 variant polypeptide having increased cell proliferativeactivity wherein; the amino acid at position 101 is selected from thegroup consisting of: Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn,Ser, Gly, Ile, Leu, or Gln; and the amino acid at position 116 isselected from the group consisting of: Lys, Leu, Pro, Thr, Met, Asp,Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; R₂ is a factorselected from the group consisting of a colony stimulating factor, acytokine, a lymphokine, an interleukin, and a hematopoietic growthfactor; and L is a linker capable of linking R₁ to R₂.
 4. The fusionprotein of claim 3, wherein said human interleukin-3 variant polypeptidehas from 1 to 14 amino acids are deleted from the N-terminus and/or from1 to 8 amino acids are deleted from the C-terminus of said humaninterluekin variant polypeptide.
 5. The fusion protein of claim 3,wherein said human interleukin-3 polypeptide has from 1 to 14 aminoacids deleted from the N-terminus and/or from 1 to 15 amino acidsdeleted from the C-terminus of said human interluekin-3 variantpolypeptide.
 6. The fusion protein of claim 2, 3, 4, or 5, wherein saidfactor is selected from the group consisting of; GM-CSF, CSF-1, G-CSF,G-CSF (Ser¹⁷), Meg-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF,flt3/ligand, human growth hormone, B-cell growth factor, B-celldifferentiation factor, eosinophil differentiation factor and stem cellfactor (SCF).
 7. The fusion protein of claim 2, 3, 4, or 5, wherein saidfactor is selected from the group consisting of: G-CSF, G-CSF (Ser¹⁷)and GM-CSF.
 8. A fusion protein consisting of a polypeptide having asequence selected from the group consisting of: R₁-L-R₂, R₂-L-R₁, R₁-R₂,R₂-L-R₁, Met-Ala-R₁-L-R₂, Met-Ala-R₂-L-R₁, Met-Ala-R₁-R₂, Met-Ala-R₂-R₁,Met-R₁-L-R₂, Met-R₂-L-R₁, Met-R₁-R₂, Met-R₂-R₁, Ala-R₁-L-R₂,Ala-R₂-L-R₁, Ala-R₁-R₂ and Ala-R₂-R₁; wherein R₁ is a humaninterleukin-3 variant polypeptide having increased cell proliferativeactivity wherein; the amino acid at position 101 is selected from thegroup consisting of: Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn,Ser, Gly, Ile, Leu, or Gln; and the amino acid at position 116 isselected from the group consisting of: Lys, Leu, Pro, Thr, Met, Asp,Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile. R₂ is a factorselected from the group consisting of a colony stimulating factor, acytokine, a lymphokine, an interleukin, and a hematopoietic growthfactor; and L is a linker capable of linking R₁ to R₂.
 9. The fusionprotein of claim 8, wherein said human interleukin-3 variant polypeptidefrom 1 to 14 amino acids are deleted from the N-terminus and/or from 1to 8 amino acids are deleted from the C-terminus of said variant hIL-3polypeptide.
 10. The fusion protein of claim 8, wherein saidbiologically active human interleukin-3 variant polypeptide from 1 to 14amino acids are deleted from the N-terminus and/or from 1 to 15 aminoacids are deleted from the C-terminus of said variant hIL-3 polypeptide.11. The fusion protein of claim 8, 9, or 10, wherein said factor isselected from the group consisting of; GM-CSF, CSF-1, G-CSF, G-CSF(Ser¹⁷), Meg-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF, flt3/ligand,human growth hormone, B-cell growth factor, B-cell differentiationfactor, eosinophil differentiation factor and stem cell factor (SCF).12. The fusion protein of claim 8, 9, or 10, wherein said factor isselected from the group consisting of: G-CSF, G-CSF (Ser¹⁷) and GM-CSF.13. A pharmaceutical composition consisting of a fusion protein of claim1, 2, 3, 4, or 5, and a pharmaceutically acceptable carrier.
 14. Apharmaceutical composition consisting of a fusion protein of claim 6 anda pharmaceutically acceptable carrier.
 15. A pharmaceutical compositionconsisting of a fusion protein of claim 7 and a pharmaceuticallyacceptable carrier.
 16. A pharmaceutical composition consisting of atherapeutically effective amount of the fusion protein of claim 8, 9, or10, and a pharmaceutically acceptable carrier.
 17. A pharmaceuticalcomposition consisting of a fusion protein of claim 11 and apharmaceutically acceptable carrier.
 18. A pharmaceutical compositionconsisting of a fusion protein of claim 12 and a pharmaceuticallyacceptable carrier.
 19. A nucleic acid molecule encoding the fusionprotein of claim
 1. 20. A nucleic acid molecule encoding the fusionprotein of claim
 2. 21. A nucleic acid molecule encoding the fusionprotein of claim
 3. 22. A nucleic acid molecule encoding the fusionprotein of claim
 4. 23. A nucleic acid molecule encoding the fusionprotein of claim
 5. 24. A nucleic acid molecule encoding the fusionprotein of claim
 6. 25. A nucleic acid molecule encoding the fusionprotein of claim
 7. 26. A nucleic acid molecule encoding the fusionprotein of claim
 8. 27. A nucleic acid molecule encoding the fusionprotein of claim
 9. 28. A nucleic acid molecule encoding the fusionprotein of claim
 10. 29. A nucleic acid molecule encoding the fusionprotein of claim
 11. 30. A nucleic acid molecule encoding the fusionprotein of claim
 12. 31. A method of producing a fusion proteincomprising: growing under suitable nutrient conditions, a host celltransformed or transfected with a replicable vector comprising saidnucleic acid molecule of claim 19, 20, 21, 22, or 23, in a mannerallowing expression of said fusion protein and recovering said fusionprotein.
 32. A method of producing a fusion protein comprising: growingunder suitable nutrient conditions, a host cell transformed ortransfected with a replicable vector comprising said nucleic acidmolecule of claim 24 in a manner allowing expression of said fusionprotein and recovering said fusion protein.
 33. A method of producing afusion protein comprising: growing under suitable nutrient conditions, ahost cell transformed or transfected with a replicable vector comprisingsaid nucleic acid molecule of claim 25 in a manner allowing expressionof said fusion protein and recovering said fusion protein.
 34. A methodof producing a fusion protein comprising; growing under suitablenutrient conditions, a host cell transformed or transfected with areplicable vector comprising said nucleic acid molecule of claim 26, 27,or 28, in a manner allowing expression of said fusion protein andrecovering said fusion protein.
 35. A method of producing a fusionprotein comprising: growing under suitable nutrient conditions, a hostcell transformed or transfected with a replicable vector comprising saidnucleic acid molecule of claim 29 in a manner allowing expression ofsaid fusion protein and recovering said fusion protein.
 36. A method ofproducing a fusion protein comprising: growing under suitable nutrientconditions, a host cell transformed or transfected with a replicablevector comprising said nucleic acid molecule of claim 30 in a mannerallowing expression of said fusion protein and recovering said fusionprotein.